Revision as of 21:00, 20 October 2019

Project

Results

qRT-PCR for the relative quantification of specific tRNA-species

Alongside with the generation of a climate-friendly medium, the goal of our project PhyCoVi was to optimize the strain Vibrio natriegens for a potential use in the biotech industry. The optimization is performed on the genomic level to increase the intracellular availability of tRNA species. As a result, the strain’s performance to express heterologous proteins is enhanced.

A method needs to be developed to quantify individual tRNA species specifically to prove the increased expression not only on the protein level. Multiple methods can be found to quantify non-coding RNA ^{1, 2} or total tRNA concentration^{3, 4}. Whereas finding a well-established method to quantify single tRNA species specifically is in vain. The only method paper was published in the journal “RNA biology” in 2015 by Honda et al.: “Four-leaf clover qRT-PCR: A convenient method for selective quantification of mature tRNA” ⁵. The authors of this paper removed the amino acid at the 3’ end followed by hybridization and ligation with a DNA/RNA hybrid stem loop creating a “four-leaf clover” shaped appearance of the tRNA ligation product. The stem loop adaptor contained a TaqMan probe binding site. During the qPCR the TaqMan probe was cut by exonuclease function of the used polymerase resulting in emission of fluorescence.

Building on the work of Honda et al. we developed a new and simplified method for relative quantification of specific tRNA species without the necessity of TaqMan probes. Instead using a DNA/RNA hybrid stem loop we used a linear DNA/RNA construct as adaptor.

The first step is to isolate RNA with a length of < 200 nt from cultured V. natriegens cells. Then, the amino acid bound to the 3’ end needs to be removed by a deacylation reaction. This results in a sticky end, where a linear RNA/DNA hybrid adaptor can be ligated, which is complementary to the 3’ end overhang. Although different tRNAs show differences in length and sequence, the last three nucleotides at the 3’ end are the same for all tRNA species. The ligated adaptor contains a binding site for the forward-primer, which is identical for all tRNAs (unspecific primer). We used T4-RNA-ligase 2 that requires ATP. For this reason, a polynucleotide kinase was necessary to carry out a phosphorylation reaction at the 5’ end.

To amplify single tRNA species specifically, we distinguished between two options. First option was using the specific tRNA primer in a reverse transcription to convert the whole tRNA pool to cDNA. Following RNase H digestion results in pure cDNA of the desired tRNA species.

Later the desired tRNA species is amplified during a qPCR by using the specific reverse primer and the unspecific adaptor primer.

During qPCR a DNA-intercalating fluorescence dye (Green DNA dye) allows for relative quantification: Green DNA dye binds to double stranded DNA and absorbs blue light and emits green light. The more double stranded DNA is generated, the higher the resulting fluorescence. And the higher the concentration of the template in the sample the faster the fluorescence exceeds the threshold. The number of cycles at which this happens is called the threshold cycle (C_t). (e.g. if sample A showed a C_t of 8 and sample B showed a C_t of 11, sample A contained 2³ = 8 times more template.)

After running a DNA gel, we noticed that the obtained amplification products did not show the expected length. This may have been a result of distinct secondary structures of the tRNA species: the reverse transcription reaction was performed at 42 °C which is the enzyme’s optimum working temperature. However, this temperature is not high enough to prevent secondary structures or to break them up. Therefore, areas with secondary structures may have been inaccessible for the reverse transcriptase resulting in shorter cDNA fragments.

For this reason, we tested a second option to amplify single tRNA species specifically. A modified polymerase together with the specific reverse primer can be used to amplify the desired tRNA species using RNA as a template. This modified polymerase works at temperatures around 65 °C and can use both RNA and DNA as a template. The reverse transcription reaction is thus not needed as a consecutive step anymore. Moreover, the modified enzyme creates the specific cDNA from RNA directly and the high temperature prevents secondary structures. The relative quantification based on C_tvalues is the same as in the option described before.

References

I. A. Babarinde, Y. Li, A. P. Hutchins (2019) Computational Methods for Mapping, Assembly and Quantification for Coding and Non-coding Transcripts, Computational and Structural Biotechnology Journal, Vol. 17, pp 628-637

D. Jacob, K. Thüring, A. Galliot, V. Marchand, A. Galvanin, A. Ciftci, K. Scharmann, M. Stock, J.‐Y. Roignant, S.A. Leidel, Y. Motorin, R. Schaffrath, R. Klassen, M. Helm (2019) Absolute Quantification of Noncoding RNA by Microscale Thermophoresis, Angewandte Chemie International Edition, Vol. 58, pp 9565 – 9569

T. S. Stenum, M. A. Sørensen, S. L. Svenningsen (2017) Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli. Journal of Visual Experiments, Issue 126, e56212

Y. Guo, A. Bosompem, S.Mohan, B. Erdogan, F.Ye, K. C. Vickers, Q. Sheng, S. Zhao, C. Li, P.-F. Su, M. Jagasia, S. A. Strickland, E. A. Griffiths, A. S. Kim (2015) Transfer RNA detection by small RNA deep sequencing and disease association with myelodysplastic syndromes, BMC Genomics, 16:727

S. Honda, M. Shigematsu, K. Morichika, A. G. Telonis, Y. Kirino (2015) Four-leaf clover qRT-PCR: A convenient method for selective quantification of mature tRNA, RNA Biology, Vol. 12, pp 501 – 508

Cloning of tRNA fragments into pSB1C3

The tRNA fragments were synthesized by IDT and amplified by PCR according to the PCR protocol (Protocol_PCR.pdf). The amplified tRNA fragments were validated via agarose gel electrophoresis (Stuttgart--Protocol_Agarose_Gel.pdf). Looking at Figure 1 all tRNA fragments showed a clear band at the desired height.

Figure 1: Amplified tRNA fragments. The tRNA fragments AGA, CGC, TGC, TCC and AGG were amplified via PCR. The PCR products were separated by agarose gel electrophoresis. A 2% agarose gel was prepared and 10 µL were loaded for each probe ((1): AGA, (2): CGC, (3): TGC, (4): TCC, (5): AGG). 3 µL of Hyberladder 1 kb Bioline were loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using MidoriGreen.

In a first step, the tRNA fragments were cloned into the pSB1C3 vector. The pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC, TCC and a combined tRNA fragment containing all 5 tRNAs were digested using the restriction enzymes XbaI and SpeI.
After purification, ( Protocol_Clean_and_Concentrate.pdf) the digested fragments were ligated using the T4 DNA ligase (see Protocol_BioBrick_Cloning.pdf). The ligated DNA fragments were transformed into DH5α ( Protocol_Transformation.pdf). The plasmid obtained from the colonies ( Protocol_Plasmid_Preparation.pdf) was digested with XbaI to gain linear Plasmid and separated by agarose gel electrophoresis ( Protocol_Agarose_Gel.pdf). Looking at Figure 2 the obtained plasmids showed no insert, only pSB1C3. Also visible is, that despite the digestion, circular, supercoiled and open circular structures of the plasmid are still present. This indicates inefficient digestion by the restriction enzymes.

Figure 2 - Cloning of tRNA fragments into pSB1C3. The pSB1C3 and the tRNA fragments were digested using the restriction enzymes XbaI and SpeI. After purification, the digested fragments were ligated using the T4 DNA ligase. The ligated DNA fragments were transformed into DH5α and subsequently prepared. The obtained plasmids were digested with XbaI before the agarose gel electrophoresis. A 1% agarose gel was prepared and 10 µL were loaded for each probe ((1): AGA, (2): AGG, (3): CGG, (4): TGC, (5): TCC, (6): Combined tRNA fragment). As a control (C) the linear pSB1C3 was loaded. 3 µL of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using GelRed.

Since the cloning showed inefficient digestion by the enzymes XbaI and SpeI, it was performed again with the enzymes EcoRI-HF and PstI. The pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC, TCC and a combined tRNA fragment were digested using the restriction enzymes EcoRI-HF and PstI.
After purification, ( Protocol_Clean_and_Concentrate.pdf) the digested fragments were ligated using the T4 DNA ligase (see Protocol_BioBrick_Cloning.pdf). The ligated DNA fragments were transformed into DH5α cells ( Protocol_Transformation.pdf). The plasmid obtained from the colonies ( Protocol_Plasmid_Preparation.pdf) was digested with EcoRI-HF to gain linear Plasmid and separated by agarose gel electrophoresis (Protocol_Agarose_Gel.pdf). The agarose gel revealed no successful cloning as all obtained plasmids showed no insert, only pSB1C3 (data not shown).

Cloning of tRNA fragments into ptRNA_backbone via BioBrick Cloning

In a first step, the self-designed linear ptRNA_backbone from IDT was ligated according to the NEB Ligation Protocol with T4 DNA Ligase (see T--Stuttgart--Blunt_End_Ligation.pdf). Afterward, the ligated ptRNA-backbone was transformed in E. coli DH5a cells (see Protocol_Transformation.pdf). Successfully transformed DH5α cells were selected on LB agar plates containing tetracycline. The next day the circular ptRNA_backbone was prepared from the colonies according to the Plasmid Preparation protocol ( Protocol_Plasmid_Preparation.pdf). The plasmid obtained from the colonies was digested with EcoRI-HF to gain linear Plasmid and separated by agarose gel electrophoresis ( Protocol_Agarose_Gel.pdf). Looking at Figure 3 all plasmids run at the desired length which corresponds to the length of the ptRNA_backbone 2159 bp.

Figure 3 -Cloning of ptRNA_backbone. The linear ptRNA_backbone fragment from IDT was ligated using the T4 DNA Ligase. The ligated ptRNA-backbone was transformed into DH5α and subsequently prepared. The obtained plasmids were digested with EcoRI-HF before the agarose gel electrophoresis. A 1% agarose gel was prepared and 10 µL were loaded for each probe ((1): ptRNA_backbone gBlock from IDT, (2): colony 2, (3): colony 3, (4): colony 4, (5): colony 5, (6): colony 6). 3 µL of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using GelRed.

The ptRNA_backbone and the previously amplified tRNA fragments AGA, AGG, CGG, TGC, TCC and a combined tRNA fragment were digested using the restriction enzymes EcoRI-HF and PstI. After purification, ( Protocol_Clean_and_Concentrate.pdf) the digested fragments were ligated using the T4 DNA ligase (see Protocol_BioBrick_Cloning.pdf). The ligated DNA fragments were transformed into DH5α ( Protocol_Transformation.pdf). The plasmid obtained from the colonies ( Protocol_Plasmid_Preparation.pdf) was digested with EcoRI-HF to gain linear Plasmid and separated by agarose gel electrophoresis ( Protocol_Agarose_Gel.pdf). Figure 4 reveals no successful cloning as all obtained Plasmids show no insert, only ptRNA_backbone.

Figure 4 - Cloning of tRNA fragments into ptRNA_backbone. The ptRNA_backbone and the tRNA fragments were digested using the restriction enzymes EcoRI-HF and PstI. After purification, the digested fragments were ligated using the T4 DNA ligase. The ligated DNA fragments were transformed into DH5α and subsequently prepared. The obtained plasmids were digested with EcoRI-HF before the agarose gel electrophoresis. A 1% agarose gel was prepared and 10 µL were loaded for each probe ((1): AGA, (2): AGG, (3): CGG, (4): TGC, (5): TCC, (6): Combined tRNA fragment). As a control (C) the linear ptRNA_backbone was loaded. 3 µL of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using GelRed.

This BioBrick cloning was repeated several times using different EcoRI-HF and PstI stocks. Following transformation in DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA_backbone. Following transformation in competent Vibrio natriegens cells also revealed no successful cloning (data not shown).

Cloning of tRNA fragments into ptRNA_backbone via Gibson Assembly

Cloning of tRNA fragments into ptRNA_backbone was also performed using Gibson Assembly. Gibson Assembly was conducted according to the protocol Gibson Assembly ( Protocol_Gibson_Assembly.pdf). The Gibson reaction was transformed into competent DH5α cells ( Protocol_Transformation.pdf). The plasmid obtained from the colonies ( Protocol_Plasmid_Preparation.pdf) was digested with EcoRI-HF to gain linear Plasmid and separated by agarose gel electrophoresis ( Protocol_Agarose_Gel.pdf). Figure 5 reveals no successful cloning as all obtained Plasmids show no insert, only ptRNA_backbone.

Figure 5 -Gibson Assembly of tRNA fragments into ptRNA_backbone. Gibson Assembly was performed according to the Gibson Assembly Protocol. The Gibson Assembly reaction was transformed into DH5α and subsequently prepared. The obtained plasmids were digested with EcoRI-HF before the agarose gel electrophoresis. A 1% agarose gel was prepared and 10 µL were loaded for each probe ((1): AGA, (2): AGG, (3): CGG, (4): TGC, (5): TCC). As a control (C) the linear ptRNA_backbone was loaded. 3 µL of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using GelRed.

Cloning of tRNA fragments into ptRNA_backbone via Gibson Assembly was repeated with another Gibson Assembly Master Mix and revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA_backbone (data not shown).

Isolation of Vibrio natriegens DSM 759 genome chr.1 and amplification of tRNAs

As an alternative to cloning of tRNA fragments provided by IDT, the tRNAs AGA, AGG, CGG, TGC and TCC were amplified from the Vibrio natriegens DSM 759 genome. Therefore, the Vibrio natriegens DSM 759 genome chr.1 was isolated according to the gDNA Extraction protocol ( gDNA_extraction.pdf). The tRNA fragments AGA, AGG, CGG, TGC, TCC were amplified from the Vibrio natriegens DSM 759 genome via PCR with appropriate primers (see Protocol_PCR.pdf). Looking at Figure 6 all tRNA fragments showed a clear band at the desired height. The tRNA fragments were extracted from the agarose gel according to the gel extraction protocol ( Protocol_Gel_Extraction.pdf).

Figure 6: Amplification of tRNAs from the Vibrio natriegens DSM 759 genome. The Vibrio natriegens DSM 759 genome chr.1 was isolated according to the Protocol gDNA Extraction. The tRNA fragments AGA, AGG, CGG, TGC, TCC were amplified from the Vibrio natriegens DSM 759 genome via PCR with appropriate primers. A 1.5 % agarose gel was prepared and 10 µL were loaded for each probe ((1): AGA, (2): AGG, (3): CGG, (4): TGC, (5): TCC). 3 µL of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using GelRed.

Cloning of tRNA fragments (amplified from Vibrio natriegens DSM 759 genome) into ptRNA_backbone via BioBrick Cloning

The ptRNA_backbone and the previously amplified tRNA fragments AGA, AGG, CGG, TGC, TCC from the Vibrio natriegens DSM 759 genome were digested using the restriction enzymes EcoRI-HF and PstI. After purification, ( Protocol_Clean_and_Concentrate.pdf) the digested fragments were ligated using the T4 DNA ligase (see Protocol_BioBrick_Cloning.pdf). The ligated DNA fragments were transformed into competent DH5α cells ( Protocol_Transformation.pdf). The plasmid obtained from the colonies ( Protocol_Plasmid_Preparation.pdf) was digested with EcoRI-HF and PstI to release inserted tRNA fragments from the ptRNA_backbone and gain linear plasmid. The DNA fragments were separated by agarose gel electrophoresis ( Protocol_Agarose_Gel.pdf). Looking at Figure 7 no insert band was visible for the obtained plasmids, only ptRNA_backbone, suggesting no successful cloning.

Figure 7: BioBrick cloning of tRNA fragments into ptRNA_backbone. The tRNA fragments were previously amplified from the Vibrio natriegens DSM 759 genome. The ptRNA_backbone and the tRNA fragments were digested using the restriction enzymes EcoRI-HF and PstI. After purification, the digested fragments were ligated using the T4 DNA ligase. The ligated DNA fragments were transformed into DH5α and subsequently prepared. The obtained plasmids were digested with EcoRI-HF and PstI before the agarose gel electrophoresis. A 1% agarose gel was prepared and 10 µL were loaded for each probe ((1): AGA, (2): AGG, (3): CGG, (4): TGC, (5): ACC). As a control (C) the linear ptRNA_backbone was loaded. 3 µL of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using GelRed.

Following transformation in competent Vibrio natriegens cells also revealed no successful cloning (data not shown).

Cloning of tRNA fragments (amplified from Vibrio natriegens DSM 759 genome) into ptRNA_backbone via NEBuilder HiFi DNA Assembly

The tRNA fragments were previously amplified from Vibrio natriegens DSM 759 genome. The NEBuilder HiFi DNA Assembly was performed according to the Protocol NEBuilder HiFi DNA Assembly (https://2019.igem.org/wiki/images/2/25/T--Stuttgart--Protocols_NEBuilder_HiFi_DNA_Assembly.pdf). Following transformation into competent DH5α cells revealed no successful cloning as no colonies were obtained. Following transformation in competent Vibrio natriegens cells also revealed no successful cloning (data not shown).

Cloning of tRNA fragments (amplified from Vibrio natriegens DSM 759 genome) into ptRNA_backbone via Gibson Assembly

Gibson Assembly was performed according to the protocol Gibson Assembly ( https://2019.igem.org/wiki/images/6/6c/T--Stuttgart--Protocol_Gibson_Assembly.pdf). Due to the orientation in the Vibrio natriegens DSM 759 genome only the tRNA fragments AGG and TGC could be used for Gibson Assembly. The Gibson reaction was transformed into competent DH5α cells ( Protocol_Transformation.pdf). The plasmid obtained from the colonies ( Protocol_Plasmid_Preparation.pdf) was digested with EcoRI-HF and PstI to release inserted tRNA fragments from the ptRNA_backbone and gain linear plasmid. The DNA fragments were separated by agarose gel electrophoresis ( Protocol_Agarose_Gel.pdf). Looking at Figure 8 no insert band was visible for the obtained plasmids, only ptRNA_backbone, suggesting no successful cloning.

Figure 8: Gibson Assembly of tRNA fragments into ptRNA_backbone. Gibson Assembly was performed according to the Gibson Assembly Protocol. The tRNA fragments were previously amplified from the Vibrio natriegens DSM 759 genome. The Gibson Assembly reaction was transformed into DH5α and subsequently prepared. The obtained plasmids were digested with EcoRI-HF and PstI before the agarose gel electrophoresis. A 1% agarose gel was prepared and 10 µL were loaded for each probe ((1): AGG colony 1, (2): AGG colony 2, (3): TGC colony 1, (4): TGC colony 2). 3 µL of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using GelRed.

Cloning of tRNA fragments into ptRNA_backbone via Gibson Assembly was repeated with another Gibson Assembly Master Mix and revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA_backbone. Following transformation of the Gibson reaction in competent Vibrio natriegens cells also revealed no successful cloning (data not shown).

Media based on algae: first tests determining important substrates

Medium based on LB

To first determine whether the extract of chlorella vulgaris was a substitute for yeast extract, LB medium and medium containing chlorella vulgaris extract instead of yeast extract were produced.

Media were produced (Protocol_media_first_experiments.pdf).
Media were inoculated with escherichia coli or vibrio natriegens.
After the over night culture, the turbidity of the tubes was observed.

Result: Growth of both bacteria was observed in both media.

Medium without tryptone

To determine whether chlorella vulgaris extract was able to substitute tryptone as a medium component, additionally, media containing different concentrations of NaCl chlorella vulgaris extract and yeast extract were prepared.

Media were produced (Protocol_media_first_experiments.pdf).
Media were inoculated with vibrio natriegens.
After the overnight culture, the turbidity of the tubes was observed.

Result: No growth was detectable in media without tryptone.

Autolysis in combination with bead-milling Results

Free amino acid estimation with rFAN assay

Samples from Experiment Cell_extraction_with_autolysis_combined_with_bead-milling.pdf were used for the analysis.

Yeast extract is mostly obtained by autolysis ¹. In autolysis cells digest their own cell compounds with their own enzymes ². The idea was to transfer this commonly used principal on algae. Therefore, C. vulgaris and C. sorokiniana were heated to 50 °C in alkaline or acidic environment for 41 h. To further crack the cell wall, both algae were treated with bead-milling afterwards. To quantify the success of cell wall disruption free amino acids were measured with rFAN-assay.

The yield of free amino acids was set into relation with the amount of biomass used in the experiment (figure 1).

Figure 1 -Autolysis and subsequent bead-milling of algae C. vulgaris and C. sorokiniana. The percentage of free amino acids [%] relates to the biomass used in the experiment.

The highest amounts of free amino acids were reached with yeast at pH 12 with 4.85 %. Both algae showed very low yield in free amino acids. The best results showed C. sorokiniana at pH 12. It is possible, that the amount of glass beads and the size of the glass beads were to little, which led to less cell wall disruption. Therefore, amino acids would have been retained within the cells. This would explain the little amounts of free amino acids achieved with this method. Also, C. vulgaris and C. sorokinia have a cell wall, in contrast to yeast ³>. Cell walls are harder to break, than a plasma membrane. This could explain the difference between the yeast samples and the algae samples. Due to the low yield in free amino acids, it was decided to investigate other methods for cell extraction of algae.

Anthrone assay to Determine Soluble Carbohydrate Concentration

Similar to the rFAN assay the anthrone assay is a method to detect free monosaccharides in a liquid. Therefor samples from the experiment Experiments_AnthroneAssay.pdf were analyzed. Hereby a calibration curve with known amounts of glucose is created (Figure 2, left side). This calibration curve creates the possibility to calculate the sugar concentration of the samples (Figure 2, right side).

Figure 2: Pictures of the anthrone calibration curve as well as the anthrone assay of samples. For the calibration curve known amounts of glucose is dissolved in water and the optical density at 620 nm is measured (left side). This can be used to determine the monosaccharide concentration of anthone treated samples which previously underwent autolysis (pH3 or pH6) with or without subsequent bead-mill treatment (RKM) (right side).

One can tell from the coloring of the samples in figure 2, that the carbohydrate concentration should differ very slightly between the samples pH3, pH6, bead mill extraction +pH3 and bead mill extraction +pH6. Due to the cloudiness of the control sample, a background corrected optical density could not be determined. Therefore, the coloring scheme served as evaluation for successful carbohydrate determination.

Hereby, bead-mill (RKM) with subsequent autolysis at pH3 was determined to be the method of choice.

References

Kim et al., “Preparation of flavor-enhancing yeast extract using a Saccharomyces cerevisiae strain with high RNA content”, Korean J Food Sci Technol, 31 (2) (1999), pp. 475-481.
T.L. Babayan, M.G. Bezrukov, “Autolysis in yeasts”, Acta Biotechnol, 5 (2) (1985), pp. 129-136.
van der Rest, M E et al. “The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.” Microbiological reviews vol. 59,2 (1995): 304-22.
Takeda, “Classification of Chlorella strains by cell wall sugar composition” Phytochemistry, vol. 27, 12, (1988), pp. 3823-3826.
[4} Takeda, “Classification of Chlorella strains by cell wall sugar composition” Phytochemistry, vol. 27, 12, (1988), pp. 3823-3826.

CDW correlation of algae Chlorella vulgaris Results

By plotting the measured optical densities against the means of the calculated cellular dry weights, a correlation was obtained. It is shown in the following figure.

Figure 1 - OD-CDW correlation of the algae Chlorella vulgaris. Mean of cellular dry weight in g/L (n=2) was plotted against the measured optical density at 750 nm. Trend line was shown in red.

The trend line in figure 1 is poorly matching the trend of the measurement points. For this reason, the correlation curve was rejected. For improvement of this experiment, measurements should be performed only by one experimenter to reduce pipetting errors or other handling mistakes. Also the measurements should be taken over a longer time period to gain more trust worthy results.

CDW-OD correlation by dilution Results

In the following table you can see the calculated cell dry weights with the corresponding optical density of the tubes. Some tubes broke during the experiment so corresponding measurements could not occur.

Table 2 -Calculated values of the cellular dry weight in g/l with the corresponding optical density measured at 750 nm.

OD	CDW[g/l]
4.87	0.98
4.41	0.88
4.44	0.91
4.02	0.8
3.92	0.82
3.52	0.68
3.57	0.71
3.03	0.57
2.65	0.49
2.8	0.5
2.43	0.48
2.56	0.46
2.29	0.4
2.28	0.4

The cellular dry weight in g/l was then plotted against the optical density measured at 750 nm. The plot with the corresponding trend line is shown in the following figure. Figure 1 - Cellular dry weight in g/l is plotted against the optical density measured at 750 nm. The linear fit is shown in blue together with its formula.

The slope of the formula further been used for fast estimation of the CDW by measuring the optical density at 750 nm.

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+            <li><a href="#algae-media-based">Media based on algae</a></li>
-                    <li><a href="#autolysis">Autolysis in combination <br/> with bead-milling Results</a></li>
+            <li>
-                    <li><a href="#cdwcorrelation">CDW correlation</a></li>
+              <a href="#autolysis"
-                    <li><a href="#cdwodcorrelation">CDW-OD correlation by dilution
+                >Autolysis in combination <br />
-                        Results</a></li>
+                with bead-milling Results</a
-                </ul>
+              >
-            </aside>
+            </li>
+            <li><a href="#cdwcorrelation">CDW correlation</a></li>
+            <li>
+              <a href="#cdwodcorrelation"
+                >CDW-OD correlation by dilution Results</a
+              >
+            </li>
+          </ul>
+        </aside>
+      </div>
+      <div class="column">
+        <div id="qPCR" class="section-container">
+          <h2 class="title is-3">
+            qRT-PCR for the relative quantification of specific tRNA-species
+          </h2>
+          <p>
+            Alongside with the generation of a climate-friendly medium, the goal
+            of our project PhyCoVi was to optimize the strain
+            <em>Vibrio natriegens</em> for a potential use in the biotech
+            industry. The optimization is performed on the genomic level to
+            increase the intracellular availability of tRNA species. As a
+            result, the strain&rsquo;s performance to express heterologous
+            proteins is enhanced.
+          </p>
+          <br />
+          <p>
+            A method needs to be developed to quantify individual tRNA species
+            specifically to prove the increased expression not only on the
+            protein level. &nbsp;Multiple methods can be found to quantify
+            non-coding RNA <sup>1, 2</sup> or total tRNA concentration<sup>
+, 4</sup
+            >. Whereas finding a well-established method to quantify single tRNA
+            species specifically is in vain. The only method paper was published
+            in the journal &ldquo;RNA biology&rdquo; in 2015 by Honda
+            <em>et al.</em>: &ldquo;Four-leaf clover qRT-PCR: A convenient
+            method for selective quantification of mature tRNA&rdquo;
+            <sup>5</sup>. The authors of this paper removed the amino acid at
+            the 3&rsquo; end followed by hybridization and ligation with a
+            DNA/RNA hybrid stem loop creating a &ldquo;four-leaf clover&rdquo;
+            shaped appearance of the tRNA ligation product. The stem loop
+            adaptor contained a TaqMan probe binding site. During the qPCR the
+            TaqMan probe was cut by exonuclease function of the used polymerase
+            resulting in emission of fluorescence.
+          </p>
+          <br />
+          <p>
+            Building on the work of Honda <em>et al.</em> we developed a new and
+            simplified method for relative quantification of specific tRNA
+            species without the necessity of TaqMan probes. Instead using a
+            DNA/RNA hybrid stem loop we used a linear DNA/RNA construct as
+            adaptor.
+          </p>
+          <br />
+          <p>
+            The first step is to isolate RNA with a length of &lt; 200 nt from
+            cultured <em>V. natriegens</em> cells. Then, the amino acid bound to
+            the 3&rsquo; end needs to be removed by a deacylation reaction. This
+            results in a sticky end, where a linear RNA/DNA hybrid adaptor can
+            be ligated, which is complementary to the 3&rsquo; end overhang.
+            Although different tRNAs show differences in length and sequence,
+            the last three nucleotides at the 3&rsquo; end are the same for all
+            tRNA species. The ligated adaptor contains a binding site for the
+            forward-primer, which is identical for all tRNAs (unspecific
+            primer). We used T4-RNA-ligase 2 that requires ATP. For this reason,
+            a polynucleotide kinase was necessary to carry out a phosphorylation
+            reaction at the 5&rsquo; end.
+          </p>
+          <br />
+          <p>
+            To amplify single tRNA species specifically, we distinguished
+            between two options. First option was using the specific tRNA primer
+            in a reverse transcription to convert the whole tRNA pool to cDNA.
+            Following RNase H digestion results in pure cDNA of the desired tRNA
+            species.
+          </p>
+          <p>
+            Later the desired tRNA species is amplified during a qPCR by using
+            the specific reverse primer and the unspecific adaptor primer.
+          </p>
+          <p>
+            During qPCR a DNA-intercalating fluorescence dye (Green DNA dye)
+            allows for relative quantification: Green DNA dye binds to double
+            stranded DNA and absorbs blue light and emits green light. The more
+            double stranded DNA is generated, the higher the resulting
+            fluorescence. And the higher the concentration of the template in
+            the sample the faster the fluorescence exceeds the threshold. The
+            number of cycles at which this happens is called the threshold cycle
+            (C<sub>t</sub>). (e.g. if sample A showed a C<sub>t</sub> of 8 and
+            sample B showed a C<sub>t</sub> of 11, sample A contained
+<sup>3</sup>&nbsp;= 8 times more template.)
+          </p>
+          <br />
+          <p>
+            After running a DNA gel, we noticed that the obtained amplification
+            products did not show the expected length. This may have been a
+            result of distinct secondary structures of the tRNA species: the
+            reverse transcription reaction was performed at 42 &deg;C which is
+            the enzyme&rsquo;s optimum working temperature. However, this
+            temperature is not high enough to prevent secondary structures or to
+            break them up. Therefore, areas with secondary structures may have
+            been inaccessible for the reverse transcriptase resulting in shorter
+            cDNA fragments.
+          </p>
+          <br />
+          <p>
+            For this reason, we tested a second option to amplify single tRNA
+            species specifically. A modified polymerase together with the
+            specific reverse primer can be used to amplify the desired tRNA
+            species using RNA as a template. This modified polymerase works at
+            temperatures around 65 &deg;C and can use both RNA and DNA as a
+            template. The reverse transcription reaction is thus not needed as a
+            consecutive step anymore. Moreover, the modified enzyme creates the
+            specific cDNA from RNA directly and the high temperature prevents
+            secondary structures. The relative quantification based on C<sub
+              >t </sub
+            >values is the same as in the option described before.
+          </p>
+          <br /><br />
+          <div class="notification">
+            <h3 class="title is-5">References</h3>
+            <ol>
+              <li>
+                I. A. Babarinde, Y. Li, A. P. Hutchins (2019) Computational
+                Methods for Mapping, Assembly and Quantification for Coding and
+                Non-coding Transcripts, Computational and Structural
+                Biotechnology Journal, Vol. 17, pp 628-637
+              </li>
+            </ol>
+            <p>&nbsp;</p>
+            <ol start="2">
+              <li>
+                D. Jacob, K. Th&uuml;ring, A. Galliot, V. Marchand, A. Galvanin,
+                A. Ciftci, K. Scharmann, M. Stock, J.‐Y. Roignant, S.A. Leidel,
+                Y. Motorin, R. Schaffrath, R. Klassen, M. Helm (2019) Absolute
+                Quantification of Noncoding RNA by Microscale Thermophoresis,
+                Angewandte Chemie International Edition, Vol. 58, pp 9565
+                &ndash; 9569
+              </li>
+            </ol>
+            <p>&nbsp;</p>
+            <ol start="3">
+              <li>
+                T. S. Stenum, M. A. S&oslash;rensen, S. L. Svenningsen (2017)
+                Quantification of the Abundance and Charging Levels of Transfer
+                RNAs in&nbsp;<em>Escherichia coli</em>.&nbsp;Journal of Visual
+                Experiments, Issue 126, e56212
+              </li>
+            </ol>
+            <p>&nbsp;</p>
+            <ol start="4">
+              <li>
+                Y. Guo, A. Bosompem, S.Mohan, B. Erdogan, F.Ye, K. C. Vickers,
+                Q. Sheng, S. Zhao, C. Li, P.-F. Su, M. Jagasia, S. A.
+                Strickland, E. A. Griffiths, A. S. Kim (2015) Transfer RNA
+                detection by small RNA deep sequencing and disease association
+                with myelodysplastic syndromes, BMC Genomics, 16:727
+              </li>
+            </ol>
+            <p>&nbsp;</p>
+            <ol start="5">
+              <li>
+                S. Honda, M. Shigematsu, K. Morichika, A. G. Telonis, Y. Kirino
+                (2015) Four-leaf clover qRT-PCR: A convenient method for
+                selective quantification of mature tRNA, RNA Biology, Vol. 12,
+                pp 501 &ndash; 508
+              </li>
+            </ol>
+          </div>
          </div>
-         <div class="column">
+         <div id="cloningtRNA" class="section-container">
-            <div id="qPCR" class="section-container">
+          <h2 class="title is-3">Cloning of tRNA fragments into pSB1C3</h2>
-                <h2 class="title is-3">qRT-PCR for the relative quantification of specific tRNA-species</h2>
+          <div class="columns">
-                <p>Alongside with the generation of a climate-friendly medium, the goal of our project PhyCoVi was to
+            <div class="column">
-                    optimize the strain <em>Vibrio natriegens</em> for a potential use in the biotech industry. The
+              <p>
-                    optimization is performed on the genomic level to increase the intracellular availability of tRNA
+                The tRNA fragments were synthesized by IDT and amplified by PCR
-                    species. As a result, the strain&rsquo;s performance to express heterologous proteins is enhanced.
+                according to the PCR protocol (<a
-                </p>
+                  href="https://2019.igem.org/wiki/images/f/f4/T--Stuttgart--Protocol_PCR.pdf"
-                <br/>
+                  >Protocol_PCR.pdf</a
-                <p>A method needs to be developed to quantify individual tRNA species specifically to prove the
+                >). The amplified tRNA fragments were validated via agarose gel
-                    increased expression not only on the protein level. &nbsp;Multiple methods can be found to quantify
+                electrophoresis (<a
-                    non-coding RNA <sup>1, 2</sup> or total tRNA concentration<sup> 3, 4</sup>. Whereas finding a
+                  href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
-                    well-established method to quantify single tRNA species specifically is in vain. The only method
+                  >Stuttgart--Protocol_Agarose_Gel.pdf</a
-                    paper was published in the journal &ldquo;RNA biology&rdquo; in 2015 by Honda <em>et al.</em>:
+                >). Looking at Figure 1 all tRNA fragments showed a clear band
-                    &ldquo;Four-leaf clover qRT-PCR: A convenient method for selective quantification of mature tRNA&rdquo;
+                at the desired height.
-                    <sup>5</sup>. The authors of this paper removed the amino acid at the 3&rsquo; end followed by
+              </p>
-                    hybridization and ligation with a DNA/RNA hybrid stem loop creating a &ldquo;four-leaf clover&rdquo;
+              <img
-                    shaped appearance of the tRNA ligation product. The stem loop adaptor contained a TaqMan probe
+                style="display: block; max-width: 500px; margin: 0 auto;"
-                    binding site. During the qPCR the TaqMan probe was cut by exonuclease function of the used
+                src="https://2019.igem.org/wiki/images/thumb/8/86/T--Stuttgart--Amplified_tRNA_fragments.png/592px-T--Stuttgart--Amplified_tRNA_fragments.png"
-                    polymerase resulting in emission of fluorescence.</p>
+              />
-                <br/>
+              <small>
-                <p>Building on the work of Honda <em>et al.</em> we developed a new and simplified method for relative
+                Figure 1: Amplified tRNA fragments. The tRNA fragments AGA, CGC,
-                    quantification of specific tRNA species without the necessity of TaqMan probes. Instead using a
+                TGC, TCC and AGG were amplified via PCR. The PCR products were
-                    DNA/RNA hybrid stem loop we used a linear DNA/RNA construct as adaptor.</p>
+                separated by agarose gel electrophoresis. A 2% agarose gel was
-                <br/>
+                prepared and 10 &micro;L were loaded for each probe ((1): AGA,
-                <p>The first step is to isolate RNA with a length of &lt; 200 nt from cultured <em>V. natriegens</em>
+                (2): CGC, (3): TGC, (4): TCC, (5): AGG). 3 &micro;L of
-                    cells. Then, the amino acid bound to the 3&rsquo; end needs to be removed by a deacylation reaction.
+                Hyberladder 1 kb Bioline were loaded as a marker (M). The gel
-                    This results in a sticky end, where a linear RNA/DNA hybrid adaptor can be ligated, which is
+                was run at 90 V for 1 hour and stained using MidoriGreen.
-                    complementary to the 3&rsquo; end overhang. Although different tRNAs show differences in length and
+              </small>
-                    sequence, the last three nucleotides at the 3&rsquo; end are the same for all tRNA species. The
+              <br />
-                    ligated adaptor contains a binding site for the forward-primer, which is identical for all tRNAs
+              <br />
-                    (unspecific primer). We used T4-RNA-ligase 2 that requires ATP. For this reason, a polynucleotide
+              <p>
-                    kinase was necessary to carry out a phosphorylation reaction at the 5&rsquo; end.</p>
+                In a first step, the tRNA fragments were cloned into the pSB1C3
-                <br/>
+                vector. The pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC,
-                <p>To amplify single tRNA species specifically, we distinguished between two options. First option was
+                TCC and a combined tRNA fragment containing all 5 tRNAs were
-                    using the specific tRNA primer in a reverse transcription to convert the whole tRNA pool to cDNA.
+                digested using the restriction enzymes XbaI and SpeI. <br />
-                    Following RNase H digestion results in pure cDNA of the desired tRNA species.</p>
+                After purification, (<a
-                <p>Later the desired tRNA species is amplified during a qPCR by using the specific reverse primer and
+                  href="https://2019.igem.org/wiki/images/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf"
-                    the unspecific adaptor primer.</p>
+                >
-                <p>During qPCR a DNA-intercalating fluorescence dye (Green DNA dye) allows for relative quantification:
+                  Protocol_Clean_and_Concentrate.pdf</a
-                    Green DNA dye binds to double stranded DNA and absorbs blue light and emits green light. The more
+                >) the digested fragments were ligated using the T4 DNA ligase
-                    double stranded DNA is generated, the higher the resulting fluorescence. And the higher the
+                (see
-                    concentration of the template in the sample the faster the fluorescence exceeds the threshold. The
+                <a
-                    number of cycles at which this happens is called the threshold cycle (C<sub>t</sub>). (e.g. if
+                  href="https://2019.igem.org/wiki/images/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf"
-                    sample A showed a C<sub>t</sub> of 8 and sample B showed a C<sub>t</sub> of 11, sample A contained 2<sup>3</sup>&nbsp;=
+                >
-times more template.)</p>
+                  Protocol_BioBrick_Cloning.pdf</a
-                <br/>
+                >). The ligated DNA fragments were transformed into DH5&alpha;
-                <p>After running a DNA gel, we noticed that the obtained amplification products did not show the
+                (<a
-                    expected length. This may have been a result of distinct secondary structures of the tRNA species:
+                  href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf"
-                    the reverse transcription reaction was performed at 42 &deg;C which is the enzyme&rsquo;s optimum
+                >
-                    working temperature. However, this temperature is not high enough to prevent secondary structures or
+                  Protocol_Transformation.pdf</a
-                    to break them up. Therefore, areas with secondary structures may have been inaccessible for the
+                >). The plasmid obtained from the colonies (<a
-                    reverse transcriptase resulting in shorter cDNA fragments.</p>
+                  href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf"
-                <br/>
+                >
-                <p>For this reason, we tested a second option to amplify single tRNA species specifically. A modified
+                  Protocol_Plasmid_Preparation.pdf</a
-                    polymerase together with the specific reverse primer can be used to amplify the desired tRNA species
+                >) was digested with XbaI to gain linear Plasmid and separated
-                    using RNA as a template. This modified polymerase works at temperatures around 65 &deg;C and can use
+                by agarose gel electrophoresis (<a
-                    both RNA and DNA as a template. The reverse transcription reaction is thus not needed as a
+                  href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
-                    consecutive step anymore. Moreover, the modified enzyme creates the specific cDNA from RNA directly
+                >
-                    and the high temperature prevents secondary structures. The relative quantification based on
+                  Protocol_Agarose_Gel.pdf</a
-                    C<sub>t </sub>values is the same as in the option described before.</p>
+                >). Looking at Figure 2 the obtained plasmids showed no insert,
-                <br><br>
+                only pSB1C3. Also visible is, that despite the digestion,
-                <div class="notification">
+                circular, supercoiled and open circular structures of the
-                    <h3 class="title is-5">References</h3>
+                plasmid are still present. This indicates inefficient digestion
-                    <ol>
+                by the restriction enzymes.
-                        <li>I. A. Babarinde, Y. Li, A. P. Hutchins (2019) Computational Methods for Mapping, Assembly
+              </p>
-                            and Quantification for Coding and Non-coding Transcripts, Computational and Structural
+              <br />
-                            Biotechnology Journal, Vol. 17, pp 628-637
+              <img
-                        </li>
+                style="display: block; max-width: 500px; margin: 0 auto;"
-                    </ol>
+                src="https://2019.igem.org/wiki/images/thumb/8/8f/T--Stuttgart--Cloning_of_tRNA_fragments_into_pSB1C3.png/722px-T--Stuttgart--Cloning_of_tRNA_fragments_into_pSB1C3.png"
-                    <p>&nbsp;</p>
+              />
-                    <ol start="2">
+              <small>
-                        <li>D. Jacob, K. Th&uuml;ring, A. Galliot, V. Marchand, A. Galvanin, A. Ciftci, K. Scharmann, M.
+                Figure 2 - Cloning of tRNA fragments into pSB1C3. The pSB1C3 and
-                            Stock, J.‐Y. Roignant, S.A. Leidel, Y. Motorin, R. Schaffrath, R. Klassen, M. Helm (2019)
+                the tRNA fragments were digested using the restriction enzymes
-                            Absolute Quantification of Noncoding RNA by Microscale Thermophoresis, Angewandte Chemie
+                XbaI and SpeI. After purification, the digested fragments were
-                            International Edition, Vol. 58, pp 9565 &ndash; 9569
+                ligated using the T4 DNA ligase. The ligated DNA fragments were
-                        </li>
+                transformed into DH5&alpha; and subsequently prepared. The
-                    </ol>
+                obtained plasmids were digested with XbaI before the agarose gel
-                    <p>&nbsp;</p>
+                electrophoresis. A 1% agarose gel was prepared and 10 &micro;L
-                    <ol start="3">
+                were loaded for each probe ((1): AGA, (2): AGG, (3): CGG, (4):
-                        <li>T. S. Stenum, M. A. S&oslash;rensen, S. L. Svenningsen (2017) Quantification of the
+                TGC, (5): TCC, (6): Combined tRNA fragment). As a control (C)
-                            Abundance and Charging Levels of Transfer RNAs in&nbsp;<em>Escherichia coli</em>.&nbsp;Journal
+                the linear pSB1C3 was loaded. 3 &micro;L of GeneRuler, 1kb Plus
-                            of Visual Experiments, Issue 126, e56212
+                DNA Ladder was loaded as a marker (M). The gel was run at 90 V
-                        </li>
+                for 1 hour and stained using GelRed.
-                    </ol>
+              </small>
-                    <p>&nbsp;</p>
+              <br />
-                    <ol start="4">
+              <br />
-                        <li>Y. Guo, A. Bosompem, S.Mohan, B. Erdogan, F.Ye, K. C. Vickers, Q. Sheng, S. Zhao, C. Li,
-                            P.-F. Su, M. Jagasia, S. A. Strickland, E. A. Griffiths, A. S. Kim (2015) Transfer RNA
-                            detection by small RNA deep sequencing and disease association with myelodysplastic
-                            syndromes, BMC Genomics, 16:727
-                        </li>
-                    </ol>
-                    <p>&nbsp;</p>
-                    <ol start="5">
-                        <li>S. Honda, M. Shigematsu, K. Morichika, A. G. Telonis, Y. Kirino (2015) Four-leaf clover
-                            qRT-PCR: A convenient method for selective quantification of mature tRNA, RNA Biology, Vol.
-, pp 501 &ndash; 508
-                        </li>
-                    </ol>
-                </div>
-            </div>
-            <div id="cloningtRNA" class="section-container">
-                <h2 class="title is-3">Cloning of tRNA fragments into pSB1C3 </h2>
-                <div class="columns">
-                    <div class="column">
-                        <p>
-                            The tRNA fragments were synthesized by IDT and amplified by PCR according to the PCR
-                            protocol
-                            (<a href="https://2019.igem.org/wiki/images/f/f4/T--Stuttgart--Protocol_PCR.pdf">Protocol_PCR.pdf</a>).
-                            The amplified tRNA fragments were validated via agarose gel electrophoresis (<a
-                                href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf">Stuttgart--Protocol_Agarose_Gel.pdf</a>).
-                            Looking at Figure 1 all tRNA fragments showed a clear band at the desired height.
-                        </p>
-                        <img style="display: block; max-width: 500px; margin: 0 auto;"
-                             src="https://2019.igem.org/wiki/images/thumb/8/86/T--Stuttgart--Amplified_tRNA_fragments.png/592px-T--Stuttgart--Amplified_tRNA_fragments.png">
-                        <small>
-                            Figure 1: Amplified tRNA fragments. The tRNA fragments AGA, CGC, TGC, TCC and AGG were
-                            amplified via PCR. The PCR products were separated by agarose gel electrophoresis. A 2%
-                            agarose gel was prepared and 10 &micro;L were loaded for each probe ((1): AGA, (2): CGC,
-                            (3): TGC, (4): TCC, (5): AGG). 3 &micro;L of Hyberladder 1 kb Bioline were loaded as a
-                            marker (M). The gel was run at 90 V for 1 hour and stained using MidoriGreen.
-                        </small>
-                        <br>
-                        <br>
-                        <p>
-                            In a first step, the tRNA fragments were cloned into the pSB1C3 vector. The pSB1C3 and the
-                            tRNA fragments AGA, AGG, CGG, TGC, TCC and a combined tRNA fragment containing all 5 tRNAs
-                            were digested using the restriction enzymes XbaI and SpeI. <br/>
-                            After purification, (<a
-                                href="https://2019.igem.org/wiki/images/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf">
-                            Protocol_Clean_and_Concentrate.pdf</a>)
-                            the digested fragments were ligated using the T4 DNA ligase (see <a
-                                href="https://2019.igem.org/wiki/images/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf">
-                            Protocol_BioBrick_Cloning.pdf</a>).
-                            The ligated DNA fragments were transformed into DH5&alpha; (<a
-                                href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf">
-                            Protocol_Transformation.pdf</a>).
-                            The plasmid obtained from the colonies (<a
-                                href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf">
-                            Protocol_Plasmid_Preparation.pdf</a>)
-                            was digested with XbaI to gain linear Plasmid and separated by agarose gel electrophoresis (<a
-                                href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf">
-                            Protocol_Agarose_Gel.pdf</a>).
-                            Looking at Figure 2 the obtained plasmids showed no insert, only pSB1C3. Also visible is,
-                            that
-                            despite the digestion, circular, supercoiled and open circular structures of the plasmid are
-                            still
-                            present. This indicates inefficient digestion by the restriction enzymes.
-                        </p>
-                        <br/>
-                        <img style="display: block; max-width: 500px; margin: 0 auto;"
-                             src="https://2019.igem.org/wiki/images/thumb/8/8f/T--Stuttgart--Cloning_of_tRNA_fragments_into_pSB1C3.png/722px-T--Stuttgart--Cloning_of_tRNA_fragments_into_pSB1C3.png">
-                        <small>
-                            Figure 2 - Cloning of tRNA fragments into pSB1C3. The pSB1C3 and the tRNA fragments were
-                            digested using the restriction enzymes XbaI and SpeI. After purification, the digested
-                            fragments were ligated using the T4 DNA ligase. The ligated DNA fragments were transformed
-                            into DH5&alpha; and subsequently prepared. The obtained plasmids were digested with XbaI
-                            before the agarose gel electrophoresis. A 1% agarose gel was prepared and 10 &micro;L were
-                            loaded for each probe ((1): AGA, (2): AGG, (3): CGG, (4): TGC, (5): TCC, (6): Combined tRNA
-                            fragment). As a control (C) the linear pSB1C3 was loaded. 3 &micro;L of GeneRuler, 1kb Plus
-                            DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using
-                            GelRed.
-                        </small>
-                        <br>
-                        <br>
-                        <p>
+              <p>
-                            Since the cloning showed inefficient digestion by the enzymes XbaI and SpeI, it was
+                Since the cloning showed inefficient digestion by the enzymes
-                            performed
+                XbaI and SpeI, it was performed again with the enzymes EcoRI-HF
-                            again with the enzymes EcoRI-HF and PstI. The pSB1C3 and the tRNA fragments AGA, AGG, CGG,
+                and PstI. The pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC,
-                            TGC, TCC and a combined tRNA fragment were digested using the restriction enzymes EcoRI-HF
+                TCC and a combined tRNA fragment were digested using the
-                            and PstI.<br>
+                restriction enzymes EcoRI-HF and PstI.<br />
-                            After purification, (<a
+                After purification, (<a
-                                href="https://2019.igem.org/wiki/images/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf">
+                  href="https://2019.igem.org/wiki/images/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf"
-                            Protocol_Clean_and_Concentrate.pdf</a>) the digested fragments were ligated using the T4
+                >
-                            DNA ligase (see <a
+                  Protocol_Clean_and_Concentrate.pdf</a
-                                href="https://2019.igem.org/wiki/images/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf">
+                >) the digested fragments were ligated using the T4 DNA ligase
-                            Protocol_BioBrick_Cloning.pdf</a>). The ligated DNA fragments were transformed into DH5&alpha;
+                (see
-                            cells (<a
+                <a
-                                href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf">
+                  href="https://2019.igem.org/wiki/images/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf"
-                            Protocol_Transformation.pdf</a>). The plasmid obtained from the colonies (<a
+                >
-                                href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf">
+                  Protocol_BioBrick_Cloning.pdf</a
-                            Protocol_Plasmid_Preparation.pdf</a>) was digested with EcoRI-HF to gain linear Plasmid
+                >). The ligated DNA fragments were transformed into DH5&alpha;
-                            and separated by agarose gel electrophoresis (<a
+                cells (<a
-                                href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf">Protocol_Agarose_Gel.pdf</a>).
+                  href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf"
-                            The agarose gel revealed no successful cloning as all obtained plasmids showed no insert,
+                >
-                            only pSB1C3 (data not shown).
+                  Protocol_Transformation.pdf</a
-                        </p>
+                >). The plasmid obtained from the colonies (<a
-                        <br>
+                  href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf"
-                        <h2 class="title is-4">Cloning of tRNA fragments into ptRNA_backbone via BioBrick Cloning</h2>
+                >
+                  Protocol_Plasmid_Preparation.pdf</a
+                >) was digested with EcoRI-HF to gain linear Plasmid and
+                separated by agarose gel electrophoresis (<a
+                  href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
+                  >Protocol_Agarose_Gel.pdf</a
+                >). The agarose gel revealed no successful cloning as all
+                obtained plasmids showed no insert, only pSB1C3 (data not
+                shown).
+              </p>
+              <br />
+              <h2 class="title is-4">
+                Cloning of tRNA fragments into ptRNA_backbone via BioBrick
+                Cloning
+              </h2>
-                        <p>
+              <p>
-                            In a first step, the self-designed linear ptRNA_backbone from IDT was ligated according to
+                In a first step, the self-designed linear ptRNA_backbone from
-                            the NEB
+                IDT was ligated according to the NEB Ligation Protocol with T4
-                            Ligation Protocol with T4 DNA Ligase (see <a
+                DNA Ligase (see
-                                href="https://2019.igem.org/wiki/images/2/2e/T--Stuttgart--Blunt_End_Ligation.pdf">
+                <a
-                            T--Stuttgart--Blunt_End_Ligation.pdf</a>).
+                  href="https://2019.igem.org/wiki/images/2/2e/T--Stuttgart--Blunt_End_Ligation.pdf"
-                            Afterward, the ligated ptRNA-backbone was transformed in <em>E. coli</em> DH5a cells (see <a
+                >
-                                href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf">
+                  T--Stuttgart--Blunt_End_Ligation.pdf</a
-                            Protocol_Transformation.pdf</a>).
+                >). Afterward, the ligated ptRNA-backbone was transformed in
-                            Successfully transformed DH5&alpha; cells were selected on LB agar plates containing
+                <em>E. coli</em> DH5a cells (see
-                            tetracycline.
+                <a
-                            The next day the circular ptRNA_backbone was prepared from the colonies according to the
+                  href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf"
-                            Plasmid
+                >
-                            Preparation protocol (<a
+                  Protocol_Transformation.pdf</a
-                                href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf">
+                >). Successfully transformed DH5&alpha; cells were selected on
-                            Protocol_Plasmid_Preparation.pdf</a>).
+                LB agar plates containing tetracycline. The next day the
-                            The plasmid obtained from the colonies was digested with EcoRI-HF to gain linear Plasmid and
+                circular ptRNA_backbone was prepared from the colonies according
-                            separated by agarose gel electrophoresis (<a
+                to the Plasmid Preparation protocol (<a
-                                href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf">
+                  href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf"
-                            Protocol_Agarose_Gel.pdf</a>).
+                >
-                            Looking at Figure 3 all plasmids run at the desired length which corresponds to the length
+                  Protocol_Plasmid_Preparation.pdf</a
-                            of the
+                >). The plasmid obtained from the colonies was digested with
-                            ptRNA_backbone 2159 bp.
+                EcoRI-HF to gain linear Plasmid and separated by agarose gel
-                        </p>
+                electrophoresis (<a
-                        <br/>
+                  href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
-                        <br/>
+                >
-                        <img style="display: block; max-width: 500px; margin: 0 auto;"
+                  Protocol_Agarose_Gel.pdf</a
-                             src="https://2019.igem.org/wiki/images/thumb/9/9f/T--Stuttgart--Cloning_of_ptRNA_backbone.png/510px-T--Stuttgart--Cloning_of_ptRNA_backbone.png">
+                >). Looking at Figure 3 all plasmids run at the desired length
-                        <small style="text-align: justify">
+                which corresponds to the length of the ptRNA_backbone 2159 bp.
-                            Figure 3 -Cloning of ptRNA_backbone. The linear ptRNA_backbone fragment from IDT was ligated
+              </p>
-                            using the T4 DNA Ligase. The ligated ptRNA-backbone was transformed into DH5&alpha; and
+              <br />
-                            subsequently prepared. The obtained plasmids were digested with EcoRI-HF before the agarose
+              <br />
-                            gel electrophoresis. A 1% agarose gel was prepared and 10 &micro;L were loaded for each
+              <img
-                            probe ((1): ptRNA_backbone gBlock from IDT, (2): colony 2, (3): colony 3, (4): colony 4,
+                style="display: block; max-width: 500px; margin: 0 auto;"
-                            (5): colony 5, (6): colony 6). 3 &micro;L of GeneRuler, 1kb Plus DNA Ladder was loaded as a
+                src="https://2019.igem.org/wiki/images/thumb/9/9f/T--Stuttgart--Cloning_of_ptRNA_backbone.png/510px-T--Stuttgart--Cloning_of_ptRNA_backbone.png"
-                            marker (M). The gel was run at 90 V for 1 hour and stained using GelRed.
+              />
-                        </small>
+              <small style="text-align: justify">
-                        <br>
+                Figure 3 -Cloning of ptRNA_backbone. The linear ptRNA_backbone
-                        <br>
+                fragment from IDT was ligated using the T4 DNA Ligase. The
-                        <p>
+                ligated ptRNA-backbone was transformed into DH5&alpha; and
-                            The ptRNA_backbone and the previously amplified tRNA fragments AGA, AGG, CGG, TGC, TCC and a
+                subsequently prepared. The obtained plasmids were digested with
-                            combined
+                EcoRI-HF before the agarose gel electrophoresis. A 1% agarose
-                            tRNA fragment were digested using the restriction enzymes EcoRI-HF and PstI. After
+                gel was prepared and 10 &micro;L were loaded for each probe
-                            purification, (<a
+                ((1): ptRNA_backbone gBlock from IDT, (2): colony 2, (3): colony
-                                href="https://2019.igem.org/wiki/images/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf">
+, (4): colony 4, (5): colony 5, (6): colony 6). 3 &micro;L of
-                            Protocol_Clean_and_Concentrate.pdf</a>)
+                GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The
-                            the digested fragments were ligated using the T4 DNA ligase (see <a
+                gel was run at 90 V for 1 hour and stained using GelRed.
-                                href="https://2019.igem.org/wiki/images/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf">
+              </small>
-                            Protocol_BioBrick_Cloning.pdf</a>).
+              <br />
-                            The ligated DNA fragments were transformed into DH5&alpha; (<a
+              <br />
-                                href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf">
+              <p>
-                            Protocol_Transformation.pdf</a>).
+                The ptRNA_backbone and the previously amplified tRNA fragments
-                            The plasmid obtained from the colonies (<a
+                AGA, AGG, CGG, TGC, TCC and a combined tRNA fragment were
-                                href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf">
+                digested using the restriction enzymes EcoRI-HF and PstI. After
-                            Protocol_Plasmid_Preparation.pdf</a>)
+                purification, (<a
-                            was digested with EcoRI-HF to gain linear Plasmid and separated by agarose gel
+                  href="https://2019.igem.org/wiki/images/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf"
-                            electrophoresis (<a
+                >
-                                href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf">
+                  Protocol_Clean_and_Concentrate.pdf</a
-                            Protocol_Agarose_Gel.pdf</a>).
+                >) the digested fragments were ligated using the T4 DNA ligase
-                            Figure 4 reveals no successful cloning as all obtained Plasmids show no insert, only
+                (see
-                            ptRNA_backbone.
+                <a
-                        </p>
+                  href="https://2019.igem.org/wiki/images/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf"
+                >
-                        <img style="display: block; max-width: 500px; margin: 0 auto;"
+                  Protocol_BioBrick_Cloning.pdf</a
-                             src="https://2019.igem.org/wiki/images/thumb/6/6a/T--Stuttgart--Cloning_of_tRNA_fragments_into_ptRNA_backbone.png/576px-T--Stuttgart--Cloning_of_tRNA_fragments_into_ptRNA_backbone.png"/>
+                >). The ligated DNA fragments were transformed into DH5&alpha;
-                        <small>
+                (<a
-                            Figure 4 - Cloning of tRNA fragments into ptRNA_backbone. The ptRNA_backbone and the tRNA
+                  href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf"
-                            fragments were digested using the restriction enzymes EcoRI-HF and PstI. After purification,
+                >
-                            the digested fragments were ligated using the T4 DNA ligase. The ligated DNA fragments were
+                  Protocol_Transformation.pdf</a
-                            transformed into DH5&alpha; and subsequently prepared. The obtained plasmids were digested
+                >). The plasmid obtained from the colonies (<a
-                            with EcoRI-HF before the agarose gel electrophoresis. A 1% agarose gel was prepared and 10
+                  href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf"
-                            &micro;L were loaded for each probe ((1): AGA, (2): AGG, (3): CGG, (4): TGC, (5): TCC, (6):
+                >
-                            Combined tRNA fragment). As a control (C) the linear ptRNA_backbone was loaded. 3 &micro;L
+                  Protocol_Plasmid_Preparation.pdf</a
-                            of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1
+                >) was digested with EcoRI-HF to gain linear Plasmid and
-                            hour and stained using GelRed.
+                separated by agarose gel electrophoresis (<a
-                        </small>
+                  href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
-                        <br/>
+                 >
-                        <p>This BioBrick cloning was repeated several times using different EcoRI-HF and PstI stocks.
+                  Protocol_Agarose_Gel.pdf</a
-                            Following transformation in DH5&alpha; revealed no successful cloning. The colonies obtained
+                 >). Figure 4 reveals no successful cloning as all obtained
-                            showed no
+                 Plasmids show no insert, only ptRNA_backbone.
-                            insert
+              </p>
-                            in an agarose gel and only ptRNA_backbone. Following transformation in competent Vibrio
-                            natriegens
-                            cells also revealed no successful cloning (data not shown).</p>
-                        <h2 class="title is-4">Cloning of tRNA fragments into ptRNA_backbone via Gibson Assembly</h2>
-                        <p>Cloning of tRNA fragments into ptRNA_backbone was also performed using Gibson Assembly.
-                            Gibson
-                            Assembly was conducted according to the protocol Gibson Assembly (<a
-                                    href="https://2019.igem.org/wiki/images/6/6c/T--Stuttgart--Protocol_Gibson_Assembly.pdf">
-                                Protocol_Gibson_Assembly.pdf</a>).
-                            The Gibson reaction was transformed into competent DH5&alpha; cells (<a
-                                    href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf">
-                                Protocol_Transformation.pdf</a>).
-                            The plasmid obtained from the colonies (<a
-                                    href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf">
-                                Protocol_Plasmid_Preparation.pdf</a>)
-                            was digested with EcoRI-HF to gain linear Plasmid and separated by agarose gel
-                            electrophoresis (<a
-                                    href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf">
-                                Protocol_Agarose_Gel.pdf</a>).
-                            Figure 5 reveals no successful cloning as all obtained Plasmids show no insert, only
-                            ptRNA_backbone.
-                        </p>
-                        <img style="display: block; max-width: 500px; margin: 0 auto;"
-                             src="https://2019.igem.org/wiki/images/thumb/8/82/T--Stuttgart--Gibson_Assembly_of_tRNA_fragments_into_ptRNA_backbone.png/527px-T--Stuttgart--Gibson_Assembly_of_tRNA_fragments_into_ptRNA_backbone.png">
-                        <small>Figure 5 -Gibson Assembly of tRNA fragments into ptRNA_backbone. Gibson Assembly was
-                            performed
-                            according to the Gibson Assembly Protocol. The Gibson Assembly reaction was transformed into
-                            DH5&alpha;
-                            and subsequently prepared. The obtained plasmids were digested with EcoRI-HF before the
-                            agarose gel
-                            electrophoresis. A 1% agarose gel was prepared and 10 &micro;L were loaded for each probe
-                            ((1): AGA,
-                            (2): AGG, (3): CGG, (4): TGC, (5): TCC). As a control (C) the linear ptRNA_backbone was
-                            loaded. 3
-                            &micro;L of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90
-                            V for 1
-                            hour and stained using GelRed.</small>
-                        <br/>
-                        <br/>
-                        <p>Cloning of tRNA fragments into ptRNA_backbone via Gibson Assembly was repeated with another
-                            Gibson
-                            Assembly Master Mix and revealed no successful cloning. The colonies obtained showed no
-                            insert in an
-                            agarose gel and only ptRNA_backbone (data not shown).</p>
-                        <h2 class="title is-4">Isolation of <em>Vibrio natriegens</em> DSM 759 genome chr.1 and
-                            amplification of
-                            tRNAs</h2>
-                        <p>As an alternative to cloning of tRNA fragments provided by IDT, the tRNAs AGA, AGG, CGG, TGC
-                            and TCC
-                            were amplified from the <em>Vibrio natriegens</em> DSM 759 genome. Therefore, the <em>Vibrio
-                                natriegens</em> DSM 759 genome chr.1 was isolated according to the gDNA Extraction
-                            protocol (<a
-                                    href="https://2019.igem.org/wiki/images/c/c0/T--Stuttgart--gDNA_extraction.pdf">
-                                gDNA_extraction.pdf</a>).
-                            The tRNA fragments AGA, AGG, CGG, TGC, TCC were amplified from the <em>Vibrio
-                                natriegens</em> DSM
-genome via PCR with appropriate primers (see <a
-                                    href="https://2019.igem.org/wiki/images/f/f4/T--Stuttgart--Protocol_PCR.pdf">
-                                Protocol_PCR.pdf</a>).
-                            Looking at Figure 6 all tRNA fragments showed a clear band at the desired height. The tRNA
-                            fragments
-                            were extracted from the agarose gel according to the gel extraction protocol (<a
-                                    href="https://2019.igem.org/wiki/images/3/32/T--Stuttgart--Protocol_Gel_Extraction.pdf">
-                                Protocol_Gel_Extraction.pdf</a>).
-                        </p>
-                        <br>
-                        <br>
-                        <img style="display: block; max-width: 500px; margin: 0 auto;"
-                             src="https://2019.igem.org/wiki/images/thumb/3/31/T--Stuttgart--Amplification_of_tRNAs_from_Vibrio_natriegens_genome.png/800px-T--Stuttgart--Amplification_of_tRNAs_from_Vibrio_natriegens_genome.png">
-                        <small>Figure 6: Amplification of tRNAs from the Vibrio natriegens DSM 759 genome. The Vibrio
-                            natriegens
-                            DSM
-genome chr.1 was isolated according to the Protocol gDNA Extraction. The tRNA fragments
-                            AGA,
-                            AGG, CGG, TGC, TCC were amplified from the Vibrio natriegens DSM 759 genome via PCR with
-                            appropriate
-                            primers. A 1.5 % agarose gel was prepared and 10 &micro;L were loaded for each probe ((1):
-                            AGA, (2):
-                            AGG, (3): CGG, (4): TGC, (5): TCC). 3 &micro;L of GeneRuler, 1kb Plus DNA Ladder was loaded
-                            as a
-                            marker (M). The gel was run at 90 V for 1 hour and stained using GelRed.</small>
-                        <br/>
-                    </div>
-                 </div>
-                <p><strong>Cloning of tRNA fragments (amplified from <em>Vibrio natriegens</em> DSM 759 genome) into
-                    ptRNA_backbone via BioBrick Cloning</strong></p>
-                <p>
-                    The ptRNA_backbone and the previously amplified tRNA fragments AGA, AGG, CGG, TGC, TCC from the <em>Vibrio
-                    natriegens</em> DSM 759 genome were digested using the restriction enzymes EcoRI-HF and PstI. After
-                    purification, (<a
-                        href="https://2019.igem.org/wiki/images/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf">
-                    Protocol_Clean_and_Concentrate.pdf</a>)
-                    the digested fragments were ligated using the T4 DNA ligase (see <a
-                        href="https://2019.igem.org/wiki/images/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf">
-                    Protocol_BioBrick_Cloning.pdf</a>).
-                    The ligated DNA fragments were transformed into competent DH5&alpha; cells (<a
-                        href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf">
-                    Protocol_Transformation.pdf</a>).
-                    The plasmid obtained from the colonies (<a
-                        href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf">
-                    Protocol_Plasmid_Preparation.pdf</a>)
-                    was digested with EcoRI-HF and PstI to release inserted tRNA fragments from the ptRNA_backbone and
-                    gain linear plasmid. The DNA fragments were separated by agarose gel electrophoresis (<a
-                        href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf">
-                    Protocol_Agarose_Gel.pdf</a>).
-                    Looking at Figure 7 no insert band was visible for the obtained plasmids, only ptRNA_backbone,
-                    suggesting no successful cloning.</p>
-                 <img style="display: block; max-width: 500px; margin: 0 auto;"
-                     src="https://2019.igem.org/wiki/images/thumb/5/5e/T--Stuttgart--Biobrick_cloning_of_tRNA_fragments_into_ptRNA_backbone.png/521px-T--Stuttgart--Biobrick_cloning_of_tRNA_fragments_into_ptRNA_backbone.png">
-                <small>
-                    Figure 7: BioBrick cloning of tRNA fragments into ptRNA_backbone. The tRNA fragments were previously
-                    amplified from the Vibrio natriegens DSM 759 genome. The ptRNA_backbone and the tRNA fragments were
-                    digested using the restriction enzymes EcoRI-HF and PstI. After purification, the digested fragments
-                    were ligated using the T4 DNA ligase. The ligated DNA fragments were transformed into DH5&alpha; and
-                    subsequently prepared. The obtained plasmids were digested with EcoRI-HF and PstI before the agarose
-                    gel electrophoresis. A 1% agarose gel was prepared and 10 &micro;L were loaded for each probe ((1):
-                    AGA, (2): AGG, (3): CGG, (4): TGC, (5): ACC). As a control (C) the linear ptRNA_backbone was loaded.
-&micro;L of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for
-hour and stained using GelRed.
-                </small>
-                <p>
-                    Following transformation in competent Vibrio natriegens cells also revealed no successful cloning
-                    (data not shown).
-                </p>
-                <br>
-                <br>
-                <h3 class="title is-4"> Cloning of tRNA fragments (amplified from <em>Vibrio natriegens</em> DSM 759
-                    genome) into ptRNA_backbone via NEBuilder HiFi DNA Assembly</h3>
-                <p>The tRNA fragments were previously amplified from <em>Vibrio natriegens</em> DSM 759 genome. The
-                    NEBuilder HiFi DNA Assembly was performed according to the Protocol NEBuilder HiFi DNA Assembly (<a
-                            href="https://2019.igem.org/wiki/images/2/25/T--Stuttgart--Protocols_NEBuilder_HiFi_DNA_Assembly.pdf">https://2019.igem.org/wiki/images/2/25/T--Stuttgart--Protocols_NEBuilder_HiFi_DNA_Assembly.pdf</a>).
-                    Following transformation into competent DH5&alpha; cells revealed no successful cloning as no
-                    colonies were obtained. Following transformation in competent <em>Vibrio natriegens</em> cells also
-                    revealed no successful cloning (data not shown).</p>
-                 <br>
-                <br>
-                <h3 class="title is-4"> Cloning of tRNA fragments (amplified from <em>Vibrio natriegens</em> DSM 759
-                    genome) into ptRNA_backbone via Gibson Assembly
-                </h3>
-                <p>Gibson Assembly was performed according to the protocol Gibson Assembly (<a
-                        href="https://2019.igem.org/wiki/images/6/6c/T--Stuttgart--Protocol_Gibson_Assembly.pdf">
-                    https://2019.igem.org/wiki/images/6/6c/T--Stuttgart--Protocol_Gibson_Assembly.pdf</a>).
-                    Due to the orientation in the <em>Vibrio natriegens</em> DSM 759 genome only the tRNA fragments AGG
-                    and TGC could be used for Gibson Assembly. The Gibson reaction was transformed into competent DH5&alpha;
-                    cells (<a href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf">
-                        Protocol_Transformation.pdf</a>).
-                    The plasmid obtained from the colonies (<a
-                            href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf">
-                        Protocol_Plasmid_Preparation.pdf</a>)
-                    was digested with EcoRI-HF and PstI to release inserted tRNA fragments from the ptRNA_backbone and
-                    gain linear plasmid. The DNA fragments were separated by agarose gel electrophoresis (<a
-                            href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf">
-                        Protocol_Agarose_Gel.pdf</a>).
-                    Looking at Figure 8 no insert band was visible for the obtained plasmids, only ptRNA_backbone,
-                    suggesting no successful cloning.
-                </p>
-                <img style="display: block; max-width: 500px; margin: 0 auto;"
-                     src="https://2019.igem.org/wiki/images/thumb/9/99/T--Stuttgart--Gibson_Assembly_of_tRNA_fragments_-2.png/512px-T--Stuttgart--Gibson_Assembly_of_tRNA_fragments_-2.png">                </p>
-                <small>
-                    Figure 8: Gibson Assembly of tRNA fragments into ptRNA_backbone. Gibson Assembly was performed
-                    according to the Gibson Assembly Protocol. The tRNA fragments were previously amplified from the
-                    Vibrio natriegens DSM 759 genome. The Gibson Assembly reaction was transformed into DH5&alpha; and
-                    subsequently prepared. The obtained plasmids were digested with EcoRI-HF and PstI before the agarose
-                    gel electrophoresis. A 1% agarose gel was prepared and 10 &micro;L were loaded for each probe ((1):
-                    AGG colony 1, (2): AGG colony 2, (3): TGC colony 1, (4): TGC colony 2). 3 &micro;L of GeneRuler, 1kb
-                    Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using
-                    GelRed.
-                </small>
-                <br/>
-                <p>
-                    Cloning of tRNA fragments into ptRNA_backbone via Gibson Assembly was repeated with another Gibson
-                    Assembly Master Mix and revealed no successful cloning. The colonies obtained showed no insert in an
-                    agarose gel and only ptRNA_backbone. Following transformation of the Gibson reaction in competent
-                    <em>Vibrio natriegens</em> cells also revealed no successful cloning (data not shown).
-                </p>
+              <img
+                style="display: block; max-width: 500px; margin: 0 auto;"
+                src="https://2019.igem.org/wiki/images/thumb/6/6a/T--Stuttgart--Cloning_of_tRNA_fragments_into_ptRNA_backbone.png/576px-T--Stuttgart--Cloning_of_tRNA_fragments_into_ptRNA_backbone.png"
+              />
+              <small>
+                Figure 4 - Cloning of tRNA fragments into ptRNA_backbone. The
+                ptRNA_backbone and the tRNA fragments were digested using the
+                restriction enzymes EcoRI-HF and PstI. After purification, the
+                digested fragments were ligated using the T4 DNA ligase. The
+                ligated DNA fragments were transformed into DH5&alpha; and
+                subsequently prepared. The obtained plasmids were digested with
+                EcoRI-HF before the agarose gel electrophoresis. A 1% agarose
+                gel was prepared and 10 &micro;L were loaded for each probe
+                ((1): AGA, (2): AGG, (3): CGG, (4): TGC, (5): TCC, (6): Combined
+                tRNA fragment). As a control (C) the linear ptRNA_backbone was
+                loaded. 3 &micro;L of GeneRuler, 1kb Plus DNA Ladder was loaded
+                as a marker (M). The gel was run at 90 V for 1 hour and stained
+                using GelRed.
+              </small>
+              <br />
+              <p>
+                This BioBrick cloning was repeated several times using different
+                EcoRI-HF and PstI stocks. Following transformation in DH5&alpha;
+                revealed no successful cloning. The colonies obtained showed no
+                insert in an agarose gel and only ptRNA_backbone. Following
+                transformation in competent Vibrio natriegens cells also
+                revealed no successful cloning (data not shown).
+              </p>
+              <br />
+              <br />
+              <h2 class="title is-4">
+                Cloning of tRNA fragments into ptRNA_backbone via Gibson
+                Assembly
+              </h2>
+              <p>
+                Cloning of tRNA fragments into ptRNA_backbone was also performed
+                using Gibson Assembly. Gibson Assembly was conducted according
+                to the protocol Gibson Assembly (<a
+                  href="https://2019.igem.org/wiki/images/6/6c/T--Stuttgart--Protocol_Gibson_Assembly.pdf"
+                >
+                  Protocol_Gibson_Assembly.pdf</a
+                >). The Gibson reaction was transformed into competent
+                DH5&alpha; cells (<a
+                  href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf"
+                >
+                  Protocol_Transformation.pdf</a
+                >). The plasmid obtained from the colonies (<a
+                  href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf"
+                >
+                  Protocol_Plasmid_Preparation.pdf</a
+                >) was digested with EcoRI-HF to gain linear Plasmid and
+                separated by agarose gel electrophoresis (<a
+                  href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
+                >
+                  Protocol_Agarose_Gel.pdf</a
+                >). Figure 5 reveals no successful cloning as all obtained
+                Plasmids show no insert, only ptRNA_backbone.
+              </p>
+              <img
+                style="display: block; max-width: 500px; margin: 0 auto;"
+                src="https://2019.igem.org/wiki/images/thumb/8/82/T--Stuttgart--Gibson_Assembly_of_tRNA_fragments_into_ptRNA_backbone.png/527px-T--Stuttgart--Gibson_Assembly_of_tRNA_fragments_into_ptRNA_backbone.png"
+              />
+              <small
+                >Figure 5 -Gibson Assembly of tRNA fragments into
+                ptRNA_backbone. Gibson Assembly was performed according to the
+                Gibson Assembly Protocol. The Gibson Assembly reaction was
+                transformed into DH5&alpha; and subsequently prepared. The
+                obtained plasmids were digested with EcoRI-HF before the agarose
+                gel electrophoresis. A 1% agarose gel was prepared and 10
+                &micro;L were loaded for each probe ((1): AGA, (2): AGG, (3):
+                CGG, (4): TGC, (5): TCC). As a control (C) the linear
+                ptRNA_backbone was loaded. 3 &micro;L of GeneRuler, 1kb Plus DNA
+                Ladder was loaded as a marker (M). The gel was run at 90 V for 1
+                hour and stained using GelRed.</small
+              >
+              <br />
+              <br />
+              <p>
+                Cloning of tRNA fragments into ptRNA_backbone via Gibson
+                Assembly was repeated with another Gibson Assembly Master Mix
+                and revealed no successful cloning. The colonies obtained showed
+                no insert in an agarose gel and only ptRNA_backbone (data not
+                shown).
+              </p>
+              <br />
+              <br />
+              <h2 class="title is-4">
+                Isolation of <em>Vibrio natriegens</em> DSM 759 genome chr.1 and
+                amplification of tRNAs
+              </h2>
+              <p>
+                As an alternative to cloning of tRNA fragments provided by IDT,
+                the tRNAs AGA, AGG, CGG, TGC and TCC were amplified from the
+                <em>Vibrio natriegens</em> DSM 759 genome. Therefore, the
+                <em>Vibrio natriegens</em> DSM 759 genome chr.1 was isolated
+                according to the gDNA Extraction protocol (<a
+                  href="https://2019.igem.org/wiki/images/c/c0/T--Stuttgart--gDNA_extraction.pdf"
+                >
+                  gDNA_extraction.pdf</a
+                >). The tRNA fragments AGA, AGG, CGG, TGC, TCC were amplified
+                from the <em>Vibrio natriegens</em> DSM 759 genome via PCR with
+                appropriate primers (see
+                <a
+                  href="https://2019.igem.org/wiki/images/f/f4/T--Stuttgart--Protocol_PCR.pdf"
+                >
+                  Protocol_PCR.pdf</a
+                >). Looking at Figure 6 all tRNA fragments showed a clear band
+                at the desired height. The tRNA fragments were extracted from
+                the agarose gel according to the gel extraction protocol (<a
+                  href="https://2019.igem.org/wiki/images/3/32/T--Stuttgart--Protocol_Gel_Extraction.pdf"
+                >
+                  Protocol_Gel_Extraction.pdf</a
+                >).
+              </p>
+              <br />
+              <br />
+              <img
+                style="display: block; max-width: 500px; margin: 0 auto;"
+                src="https://2019.igem.org/wiki/images/thumb/3/31/T--Stuttgart--Amplification_of_tRNAs_from_Vibrio_natriegens_genome.png/800px-T--Stuttgart--Amplification_of_tRNAs_from_Vibrio_natriegens_genome.png"
+              />
+              <small
+                >Figure 6: Amplification of tRNAs from the Vibrio natriegens DSM
+genome. The Vibrio natriegens DSM 759 genome chr.1 was
+                isolated according to the Protocol gDNA Extraction. The tRNA
+                fragments AGA, AGG, CGG, TGC, TCC were amplified from the Vibrio
+                natriegens DSM 759 genome via PCR with appropriate primers. A
+.5 % agarose gel was prepared and 10 &micro;L were loaded for
+                each probe ((1): AGA, (2): AGG, (3): CGG, (4): TGC, (5): TCC). 3
+                &micro;L of GeneRuler, 1kb Plus DNA Ladder was loaded as a
+                marker (M). The gel was run at 90 V for 1 hour and stained using
+                GelRed.</small
+              >
+              <br />
              </div>
-            <br/><br/>
+          </div>
+          <p>
+            <strong
+              >Cloning of tRNA fragments (amplified from
+              <em>Vibrio natriegens</em> DSM 759 genome) into ptRNA_backbone via
+              BioBrick Cloning</strong
+            >
+          </p>
+          <p>
+            The ptRNA_backbone and the previously amplified tRNA fragments AGA,
+            AGG, CGG, TGC, TCC from the <em>Vibrio natriegens</em> DSM 759
+            genome were digested using the restriction enzymes EcoRI-HF and
+            PstI. After purification, (<a
+              href="https://2019.igem.org/wiki/images/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf"
+            >
+              Protocol_Clean_and_Concentrate.pdf</a
+            >) the digested fragments were ligated using the T4 DNA ligase (see
+            <a
+              href="https://2019.igem.org/wiki/images/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf"
+            >
+              Protocol_BioBrick_Cloning.pdf</a
+            >). The ligated DNA fragments were transformed into competent
+            DH5&alpha; cells (<a
+              href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf"
+            >
+              Protocol_Transformation.pdf</a
+            >). The plasmid obtained from the colonies (<a
+              href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf"
+            >
+              Protocol_Plasmid_Preparation.pdf</a
+            >) was digested with EcoRI-HF and PstI to release inserted tRNA
+            fragments from the ptRNA_backbone and gain linear plasmid. The DNA
+            fragments were separated by agarose gel electrophoresis (<a
+              href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
+            >
+              Protocol_Agarose_Gel.pdf</a
+            >). Looking at Figure 7 no insert band was visible for the obtained
+            plasmids, only ptRNA_backbone, suggesting no successful cloning.
+          </p>
+          <img
+            style="display: block; max-width: 500px; margin: 0 auto;"
+            src="https://2019.igem.org/wiki/images/thumb/5/5e/T--Stuttgart--Biobrick_cloning_of_tRNA_fragments_into_ptRNA_backbone.png/521px-T--Stuttgart--Biobrick_cloning_of_tRNA_fragments_into_ptRNA_backbone.png"
+          />
+          <small>
+            Figure 7: BioBrick cloning of tRNA fragments into ptRNA_backbone.
+            The tRNA fragments were previously amplified from the Vibrio
+            natriegens DSM 759 genome. The ptRNA_backbone and the tRNA fragments
+            were digested using the restriction enzymes EcoRI-HF and PstI. After
+            purification, the digested fragments were ligated using the T4 DNA
+            ligase. The ligated DNA fragments were transformed into DH5&alpha;
+            and subsequently prepared. The obtained plasmids were digested with
+            EcoRI-HF and PstI before the agarose gel electrophoresis. A 1%
+            agarose gel was prepared and 10 &micro;L were loaded for each probe
+            ((1): AGA, (2): AGG, (3): CGG, (4): TGC, (5): ACC). As a control (C)
+            the linear ptRNA_backbone was loaded. 3 &micro;L of GeneRuler, 1kb
+            Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V
+            for 1 hour and stained using GelRed.
+          </small>
+          <p>
+            Following transformation in competent Vibrio natriegens cells also
+            revealed no successful cloning (data not shown).
+          </p>
+          <br />
+          <br />
+          <h3 class="title is-4">
+            Cloning of tRNA fragments (amplified from
+            <em>Vibrio natriegens</em> DSM 759 genome) into ptRNA_backbone via
+            NEBuilder HiFi DNA Assembly
+          </h3>
+          <p>
+            The tRNA fragments were previously amplified from
+            <em>Vibrio natriegens</em> DSM 759 genome. The NEBuilder HiFi DNA
+            Assembly was performed according to the Protocol NEBuilder HiFi DNA
+            Assembly (<a
+              href="https://2019.igem.org/wiki/images/2/25/T--Stuttgart--Protocols_NEBuilder_HiFi_DNA_Assembly.pdf"
+              >https://2019.igem.org/wiki/images/2/25/T--Stuttgart--Protocols_NEBuilder_HiFi_DNA_Assembly.pdf</a
+            >). Following transformation into competent DH5&alpha; cells
+            revealed no successful cloning as no colonies were obtained.
+            Following transformation in competent
+            <em>Vibrio natriegens</em> cells also revealed no successful cloning
+            (data not shown).
+          </p>
-            <div id="algae-media-based" class="section-container">
+          <br />
-                <h2 class="title is-3">Media based on algae: first tests determining important substrates</h2>
+          <br />
-                <h3 class="title is-4">Medium based on LB</h3>
+          <h3 class="title is-4">
-                <p>To first determine whether the extract of <em>chlorella vulgaris </em>was a substitute for yeast
+            Cloning of tRNA fragments (amplified from
-                    extract, LB medium and medium containing <em>chlorella vulgaris</em> extract instead of yeast
+            <em>Vibrio natriegens</em> DSM 759 genome) into ptRNA_backbone via
-                    extract were produced.</p>
+             Gibson Assembly
-                <ol>
+          </h3>
-                    <li>Media were produced (<a
-                            href="https://2019.igem.org/File:T--Stuttgart--Protocol_media_first_experiments.pdf">Protocol_media_first_experiments.pdf</a>).
-                    </li>
-                    <li>Media were inoculated with <em>escherichia coli</em> or <em>vibrio natriegens.</em></li>
-                    <li>After the over night culture, the turbidity of the tubes was observed.</li>
-                </ol>
-                <br/>
-                <p>Result: Growth of both bacteria was observed in both media.</p>
-                <br/>
-                <h3 class="title is-4">Medium without tryptone</h3>
-                <p>To determine whether <em>chlorella vulgaris</em> extract was able to substitute tryptone as a medium
-                    component, additionally, media containing different concentrations of NaCl <em>chlorella
-                        vulgaris</em> extract and yeast extract were prepared.</p>
-                <ol>
-                    <li>Media were produced (<a
-                            href="https://2019.igem.org/File:T--Stuttgart--Protocol_media_first_experiments.pdf">Protocol_media_first_experiments.pdf</a>).
-                    </li>
-                    <li>Media were inoculated with <em>vibrio natriegens.</em></li>
-                    <li>After the overnight culture, the turbidity of the tubes was observed.</li>
-                </ol>
-                <p>Result: No growth was detectable in media without tryptone.</p>
-             </div>
-            <br/><br/>
-            <div id="autolysis" class="section-container">
-                <h2 class="title is-3">Autolysis in combination with bead-milling Results</h2>
-                <h3 class="title is-4">Free amino acid estimation with rFAN assay</h3>
-                <p>
-                    Samples from Experiment <a target="_blank"
-                                               href="https://2019.igem.org/wiki/images/e/ec/T--Stuttgart--Protocol_Cell_extraction_with_autolysis_combined_with_bead-milling.pdf">Cell_extraction_with_autolysis_combined_with_bead-milling.pdf</a>
-                    were used for the analysis.
-                </p>
-                <br/>
-                <p>Yeast extract is mostly obtained by autolysis <sup>1</sup>. In autolysis cells digest their own cell
-                    compounds
-                    with their own enzymes <sup>2</sup>. The idea was to transfer this commonly used principal on algae.
-                    Therefore,
-                    <em>C. vulgaris </em>and <em>C. sorokiniana </em>were heated to 50&nbsp;&deg;C in alkaline or acidic
-                    environment for 41&nbsp;h. To further crack the cell wall, both algae were treated with bead-milling
-                    afterwards. To quantify the success of cell wall disruption free amino acids were measured with
-                    rFAN-assay.</p>
-                <br/>
-                <p>The yield of free amino acids was set into relation with the amount of biomass used in the experiment
-                    (figure 1).</p>
-                <div class="has-text-centered" style="width: 75%; margin:0 auto;">
+          <p>
-                    <canvas id="autolysis-figure-1"></canvas>
+            Gibson Assembly was performed according to the protocol Gibson
-                </div>
+            Assembly (<a
+              href="https://2019.igem.org/wiki/images/6/6c/T--Stuttgart--Protocol_Gibson_Assembly.pdf"
+            >
+              https://2019.igem.org/wiki/images/6/6c/T--Stuttgart--Protocol_Gibson_Assembly.pdf</a
+            >). Due to the orientation in the <em>Vibrio natriegens</em> DSM 759
+            genome only the tRNA fragments AGG and TGC could be used for Gibson
+            Assembly. The Gibson reaction was transformed into competent
+            DH5&alpha; cells (<a
+              href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf"
+            >
+              Protocol_Transformation.pdf</a
+            >). The plasmid obtained from the colonies (<a
+              href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf"
+            >
+              Protocol_Plasmid_Preparation.pdf</a
+            >) was digested with EcoRI-HF and PstI to release inserted tRNA
+            fragments from the ptRNA_backbone and gain linear plasmid. The DNA
+            fragments were separated by agarose gel electrophoresis (<a
+              href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
+            >
+              Protocol_Agarose_Gel.pdf</a
+            >). Looking at Figure 8 no insert band was visible for the obtained
+            plasmids, only ptRNA_backbone, suggesting no successful cloning.
+          </p>
+          <img
+            style="display: block; max-width: 500px; margin: 0 auto;"
+            src="https://2019.igem.org/wiki/images/thumb/9/99/T--Stuttgart--Gibson_Assembly_of_tRNA_fragments_-2.png/512px-T--Stuttgart--Gibson_Assembly_of_tRNA_fragments_-2.png"
+          />
+          <small>
+            Figure 8: Gibson Assembly of tRNA fragments into ptRNA_backbone.
+            Gibson Assembly was performed according to the Gibson Assembly
+            Protocol. The tRNA fragments were previously amplified from the
+            Vibrio natriegens DSM 759 genome. The Gibson Assembly reaction was
+            transformed into DH5&alpha; and subsequently prepared. The obtained
+            plasmids were digested with EcoRI-HF and PstI before the agarose gel
+            electrophoresis. A 1% agarose gel was prepared and 10 &micro;L were
+            loaded for each probe ((1): AGG colony 1, (2): AGG colony 2, (3):
+            TGC colony 1, (4): TGC colony 2). 3 &micro;L of GeneRuler, 1kb Plus
+            DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1
+            hour and stained using GelRed.
+          </small>
+          <br />
+          <p>
+            Cloning of tRNA fragments into ptRNA_backbone via Gibson Assembly
+            was repeated with another Gibson Assembly Master Mix and revealed no
+            successful cloning. The colonies obtained showed no insert in an
+            agarose gel and only ptRNA_backbone. Following transformation of the
+            Gibson reaction in competent
+            <em>Vibrio natriegens</em> cells also revealed no successful cloning
+            (data not shown).
+          </p>
+        </div>
+        <br /><br />
+        <div id="algae-media-based" class="section-container">
+          <h2 class="title is-3">
+            Media based on algae: first tests determining important substrates
+          </h2>
+          <h3 class="title is-4">Medium based on LB</h3>
+          <p>
+            To first determine whether the extract of
+            <em>chlorella vulgaris </em>was a substitute for yeast extract, LB
+            medium and medium containing <em>chlorella vulgaris</em> extract
+            instead of yeast extract were produced.
+          </p>
+          <ol>
+            <li>
+              Media were produced (<a
+                href="https://2019.igem.org/File:T--Stuttgart--Protocol_media_first_experiments.pdf"
+                >Protocol_media_first_experiments.pdf</a
+              >).
+            </li>
+            <li>
+              Media were inoculated with <em>escherichia coli</em> or
+              <em>vibrio natriegens.</em>
+            </li>
+            <li>
+              After the over night culture, the turbidity of the tubes was
+              observed.
+            </li>
+          </ol>
+          <br />
+          <p>Result: Growth of both bacteria was observed in both media.</p>
+          <br />
+          <h3 class="title is-4">Medium without tryptone</h3>
+          <p>
+            To determine whether <em>chlorella vulgaris</em> extract was able to
+            substitute tryptone as a medium component, additionally, media
+            containing different concentrations of NaCl
+            <em>chlorella vulgaris</em> extract and yeast extract were prepared.
+          </p>
+          <ol>
+            <li>
+              Media were produced (<a
+                href="https://2019.igem.org/File:T--Stuttgart--Protocol_media_first_experiments.pdf"
+                >Protocol_media_first_experiments.pdf</a
+              >).
+            </li>
+            <li>Media were inoculated with <em>vibrio natriegens.</em></li>
+            <li>
+              After the overnight culture, the turbidity of the tubes was
+              observed.
+            </li>
+          </ol>
+          <p>Result: No growth was detectable in media without tryptone.</p>
+        </div>
+        <br /><br />
+        <div id="autolysis" class="section-container">
+          <h2 class="title is-3">
+            Autolysis in combination with bead-milling Results
+          </h2>
+          <h3 class="title is-4">Free amino acid estimation with rFAN assay</h3>
+          <p>
+            Samples from Experiment
+            <a
+              target="_blank"
+              href="https://2019.igem.org/wiki/images/e/ec/T--Stuttgart--Protocol_Cell_extraction_with_autolysis_combined_with_bead-milling.pdf"
+              >Cell_extraction_with_autolysis_combined_with_bead-milling.pdf</a
+            >
+            were used for the analysis.
+          </p>
+          <br />
+          <p>
+            Yeast extract is mostly obtained by autolysis <sup>1</sup>. In
+            autolysis cells digest their own cell compounds with their own
+            enzymes <sup>2</sup>. The idea was to transfer this commonly used
+            principal on algae. Therefore, <em>C. vulgaris </em>and
+            <em>C. sorokiniana </em>were heated to 50&nbsp;&deg;C in alkaline or
+            acidic environment for 41&nbsp;h. To further crack the cell wall,
+            both algae were treated with bead-milling afterwards. To quantify
+            the success of cell wall disruption free amino acids were measured
+            with rFAN-assay.
+          </p>
+          <br />
+          <p>
+            The yield of free amino acids was set into relation with the amount
+            of biomass used in the experiment (figure 1).
+          </p>
-                <script>
+          <div class="has-text-centered" style="width: 75%; margin:0 auto;">
-                    window.onload = function () {
+            <canvas id="autolysis-figure-1"></canvas>
-                        var autolysisfigure1 = document.getElementById('autolysis-figure-1').getContext('2d');
+          </div>
-                        window.myBar = new Chart(autolysisfigure1, {
-                            type: 'bar',
-                            data: {
-                                labels: ["yeast pH3",
-                                    "yeast pH12",
-                                    "C.vulgaris pH3",
-                                    "C.vulgaris pH12",
-                                    "C.sorokeniana pH3",
-                                    "C.sorokeniana pH12",
-                                ],
-                                datasets: [
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-                                        label: "Population (millions)",
-                                        data: [3.132222386,
-.852523798,
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-.043678259,
-.087077261,
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-                                    }]
-                            },
+          <script>
-                            options: {
+            window.onload = function() {
-                                responsive: true,
+              var autolysisfigure1 = document
-                                legend: {
+                .getElementById("autolysis-figure-1")
-                                    position: 'top',
+                .getContext("2d");
-                                }, scales: {
+              window.myBar = new Chart(autolysisfigure1, {
-                                    yAxes: [
+                type: "bar",
-                                        {
+                data: {
-                                            scaleLabel: {
+                  labels: [
-                                                display: true,
+                    "yeast pH3",
-                                                labelString: "percentage of free amio acids [%]"
+                    "yeast pH12",
-                                            }
+                    "C.vulgaris pH3",
-                                        }
+                    "C.vulgaris pH12",
-                                    ]
+                    "C.sorokeniana pH3",
-                                },
+                    "C.sorokeniana pH12"
-                            }
+                  ],
-                        });
+                  datasets: [
+                    {
+                      backgroundColor: [
+                        "#3e95cd",
+                        "#3e95cd",
+                        "#3e95cd",
+                        "#3e95cd",
+                        "#3e95cd",
+                        "#3e95cd"
+                      ],
+                      label: "Population (millions)",
+                      data: [
+.132222386,
+.852523798,
+.021842627,
+.037566219,
+.043678259,
+.087077261
+                      ]
+                    }
+                  ]
+                },
+                options: {
+                  responsive: true,
+                  legend: {
+                    position: "top"
+                  },
+                  scales: {
+                    yAxes: [
+                      {
+                        scaleLabel: {
+                          display: true,
+                          labelString: "percentage of free amio acids [%]"
+                        }
+                      }
+                    ]
+                  }
+                }
+              });
+            };
+          </script>
-                    };
+          <small
-                </script>
+            >Figure 1 -Autolysis and subsequent bead-milling of algae C.
+            vulgaris and C. sorokiniana. The percentage of free amino acids [%]
+            relates to the biomass used in the experiment.</small
+          >
+          <br />
+          <p>
+            The highest amounts of free amino acids were reached with yeast at
+            pH 12 with 4.85&nbsp;%. Both algae showed very low yield in free
+            amino acids. The best results showed <em>C. sorokiniana</em> at
+            pH&nbsp;12. It is possible, that the amount of glass beads and the
+            size of the glass beads were to little, which led to less cell wall
+            disruption. Therefore, amino acids would have been retained within
+            the cells. This would explain the little amounts of free amino acids
+            achieved with this method. Also, <em>C. vulgaris </em>and
+            <em>C. sorokinia </em>have a cell wall, in contrast to yeast
+            <sup>3</sup>>. Cell walls are harder to break, than a plasma
+            membrane. This could explain the difference between the yeast
+            samples and the algae samples. Due to the low yield in free amino
+            acids, it was decided to investigate other methods for cell
+            extraction of algae.
+          </p>
+          <br />
+          <br />
+          <h3 class="title is-4">
+            Anthrone assay to Determine Soluble Carbohydrate Concentration
+          </h3>
+          <p>
+            Similar to the rFAN assay the anthrone assay is a method to detect
+            free monosaccharides in a liquid. Therefor samples from the
+            experiment
+            <a
+              href="https://2019.igem.org/wiki/images/f/f3/T--Stuttgart--Experiments_AnthroneAssay.pdf"
+              >Experiments_AnthroneAssay.pdf</a
+            >
+            were analyzed. Hereby a calibration curve with known amounts of
+            glucose is created (Figure 2, left side). This calibration curve
+            creates the possibility to calculate the sugar concentration of the
+            samples (Figure 2, right side).
+          </p>
-                <small>Figure 1 -Autolysis and subsequent bead-milling of algae C. vulgaris and C. sorokiniana. The
+          <img
-                    percentage of free amino acids [%] relates to the biomass used in the experiment.</small>
+            style="display: block; margin:0 auto;"
-                <br>
+            src="https://2019.igem.org/wiki/images/thumb/2/2f/T--Stuttgart--FigureAutolysis_in_combination_with_bead-milling_Results2.png/800px-T--Stuttgart--FigureAutolysis_in_combination_with_bead-milling_Results2.png"
-                <p>The highest amounts of free amino acids were reached with yeast at pH 12 with 4.85&nbsp;%. Both algae
+          />
-                    showed very low yield in free amino acids. The best results showed <em>C. sorokiniana</em> at pH&nbsp;12.
+          <small
-                    It is possible, that the amount of glass beads and the size of the glass beads were to little, which
+            >Figure 2: Pictures of the anthrone calibration curve as well as the
-                    led
+            anthrone assay of samples. For the calibration curve known amounts
-                    to less cell wall disruption. Therefore, amino acids would have been retained within the cells. This
+            of glucose is dissolved in water and the optical density at 620 nm
-                    would explain the little amounts of free amino acids achieved with this method. Also, <em>C.
+            is measured (left side). This can be used to determine the
-                        vulgaris </em>and <em>C. sorokinia </em>have a cell wall, in contrast to yeast <sup>3</sup>>.
+            monosaccharide concentration of anthone treated samples which
-                    Cell walls
+            previously underwent autolysis (pH3 or pH6) with or without
-                    are
+            subsequent bead-mill treatment (RKM) (right side).</small
-                    harder to break, than a plasma membrane. This could explain the difference between the yeast samples
+          >
-                    and
-                    the algae samples. Due to the low yield in free amino acids, it was decided to investigate other
-                    methods
-                    for cell extraction of algae.</p>
-                <br>
-                <br>
-                <h3 class="title is-4">Anthrone assay to Determine Soluble Carbohydrate Concentration</h3>
-                <p>Similar to the rFAN assay the anthrone assay is a method to detect free monosaccharides in a liquid.
-                    Therefor samples from the experiment <a
-                            href="https://2019.igem.org/wiki/images/f/f3/T--Stuttgart--Experiments_AnthroneAssay.pdf">Experiments_AnthroneAssay.pdf</a>
-                    were analyzed. Hereby a calibration curve with known amounts of glucose is created (Figure 2, left
-                    side). This calibration curve creates the possibility to calculate the sugar concentration of the
-                    samples (Figure 2, right side).</p>
-                 <img style="display: block; margin:0 auto;"
+          <br />
-                     src="https://2019.igem.org/wiki/images/thumb/2/2f/T--Stuttgart--FigureAutolysis_in_combination_with_bead-milling_Results2.png/800px-T--Stuttgart--FigureAutolysis_in_combination_with_bead-milling_Results2.png">
+          <br />
-                <small>Figure 2: Pictures of the anthrone calibration curve as well as the anthrone assay of samples.
+          <p>
-                    For the calibration curve known amounts of glucose is dissolved in water and the optical density at
+            One can tell from the coloring of the samples in figure 2, that the
-nm is measured (left side). This can be used to determine the monosaccharide concentration of
+            carbohydrate concentration should differ very slightly between the
-                    anthone treated samples which previously underwent autolysis (pH3 or pH6) with or without subsequent
+            samples pH3, pH6, bead mill extraction +pH3 and bead mill extraction
-                    bead-mill treatment (RKM) (right side).</small>
+            +pH6. Due to the cloudiness of the control sample, a background
+            corrected optical density could not be determined. Therefore, the
+            coloring scheme served as evaluation for successful carbohydrate
+            determination.
+          </p>
+          <p>
+            Hereby, bead-mill (RKM) with subsequent autolysis at pH3 was
+            determined to be the method of choice.
+          </p>
+          <br /><br />
+          <div class="notification">
+            <h3 class="title is-5">References</h3>
+            <ol>
+              <li>
+                 Kim et al., &ldquo;Preparation of flavor-enhancing yeast extract
+                using a Saccharomyces cerevisiae strain with high RNA
+                content&rdquo;, Korean J Food Sci Technol, 31 (2) (1999), pp.
+-481.
+              </li>
+              <li>
+                T.L. Babayan, M.G. Bezrukov, &ldquo;Autolysis in yeasts&rdquo;,
+                Acta Biotechnol, 5 (2) (1985), pp. 129-136.
+              </li>
+              <li>
+                van der Rest, M E et al. &ldquo;The plasma membrane of
+                Saccharomyces cerevisiae: structure, function, and
+                biogenesis.&rdquo; Microbiological reviews vol. 59,2 (1995):
+-22.
+              </li>
+              <li>
+                Takeda, &ldquo;Classification of Chlorella strains by cell wall
+                sugar composition&rdquo; Phytochemistry, vol. 27, 12, (1988),
+                pp. 3823-3826.
+              </li>
+              <li>
+                [4} Takeda, &ldquo;Classification of Chlorella strains by cell
+                wall sugar composition&rdquo; Phytochemistry, vol. 27, 12,
+                (1988), pp. 3823-3826.
+              </li>
+            </ol>
+          </div>
+        </div>
+        <br /><br />
+        <div id="cdwcorrelation" class="section-container">
+          <h2 class="title is-3">
+            CDW correlation of algae <em>Chlorella vulgaris</em> Results
+          </h2>
+          <p>
+            By plotting the measured optical densities against the means of the
+            calculated cellular dry weights, a correlation was obtained. It is
+            shown in the following figure.
+          </p>
+          <br />
+          <img
+            style="display: block; max-width: 500px; margin: 0 auto;"
+            src="https://2019.igem.org/wiki/images/a/ad/T--Stuttgart--FigureOD-CDW_correlation_of_algae_Chlorella_vulgaris_Results1.png"
+          />
+          <small
+            >Figure 1 - OD-CDW correlation of the algae
+            <em>Chlorella vulgaris</em>. Mean of cellular dry weight in g/L
+            (n=2) was plotted against the measured optical density at 750 nm.
+            Trend line was shown in red.
+          </small>
+          <br />
+          <br />
+          <br />
+          <p>
+            The trend line in figure 1 is poorly matching the trend of the
+            measurement points. For this reason, the correlation curve was
+            rejected. For improvement of this experiment, measurements should be
+            performed only by one experimenter to reduce pipetting errors or
+            other handling mistakes. Also the measurements should be taken over
+            a longer time period to gain more trust worthy results.
+          </p>
+          <br />
+        </div>
+        <br /><br />
+        <div id="cdwodcorrelation" class="section-container">
+          <h2 class="title is-3">CDW-OD correlation by dilution Results</h2>
+          In the following table you can see the calculated cell dry weights
+          with the corresponding optical density of the tubes. Some tubes broke
+          during the experiment so corresponding measurements could not occur.
+          <br />
+          <br />
+          <div class="columns">
+            <div class="column">
+              <small
+                >Table 2 -Calculated values of the cellular dry weight in g/l
+                with the corresponding optical density measured at 750 nm.
+              </small>
-                 <br>
+              <table class="table is-fullwidth">
-                 <br>
+                 <thead>
-                 <p>One can tell from the coloring of the samples in figure 2, that the carbohydrate concentration should
+                  <tr>
-                     differ very slightly between the samples pH3, pH6, bead mill extraction +pH3 and bead mill
+                    <th>OD</th>
-                     extraction +pH6. Due to the cloudiness of the control sample, a background corrected optical density
+                    <th>CDW[g/l]</th>
-                     could not be determined. Therefore, the coloring scheme served as evaluation for successful
+                  </tr>
-                     carbohydrate determination.</p>
+                 </thead>
-                <p>Hereby, bead-mill (RKM) with subsequent autolysis at pH3 was determined to be the method of
+                 <tbody>
-                     choice.</p>
+                  <tr>
-                <br><br>
+                     <td>4.87</td>
-                <div class="notification">
+                     <td>0.98</td>
-                     <h3 class="title is-5">References</h3>
+                  </tr>
-                     <ol>
+                  <tr>
-                        <li>Kim et al., &ldquo;Preparation of flavor-enhancing yeast extract using a Saccharomyces
+                     <td>4.41</td>
-                            cerevisiae strain with high RNA content&rdquo;, Korean J Food Sci Technol, 31 (2) (1999),
+                     <td>0.88</td>
-                            pp.
+                  </tr>
--481.
+                  <tr>
-                        </li>
+                     <td>4.44</td>
-                        <li>T.L. Babayan, M.G. Bezrukov, &ldquo;Autolysis in yeasts&rdquo;, Acta Biotechnol, 5 (2)
+                    <td>0.91</td>
-                            (1985), pp. 129-136.
+                  </tr>
-                        </li>
+                  <tr>
-                        <li>van der Rest, M E et al. &ldquo;The plasma membrane of Saccharomyces cerevisiae: structure,
+                     <td>4.02</td>
-                            function, and biogenesis.&rdquo; Microbiological reviews vol. 59,2 (1995): 304-22.
+                     <td>0.8</td>
-                        </li>
+                  </tr>
-                        <li>Takeda, &ldquo;Classification of Chlorella strains by cell wall sugar composition&rdquo;
+                  <tr>
-                            Phytochemistry, vol. 27, 12, (1988), pp. 3823-3826.
+                    <td>3.92</td>
-                        </li>
+                    <td>0.82</td>
-                        <li>[4} Takeda, &ldquo;Classification of Chlorella strains by cell wall sugar composition&rdquo;
+                  </tr>
-                            Phytochemistry, vol. 27, 12, (1988), pp. 3823-3826.
+                  <tr>
-                        </li>
+                    <td>3.52</td>
-                     </ol>
+                    <td>0.68</td>
-                 </div>
+                  </tr>
+                  <tr>
+                    <td>3.57</td>
+                    <td>0.71</td>
+                  </tr>
+                  <tr>
+                    <td>3.03</td>
+                    <td>0.57</td>
+                  </tr>
+                  <tr>
+                    <td>2.65</td>
+                    <td>0.49</td>
+                  </tr>
+                  <tr>
+                    <td>2.8</td>
+                    <td>0.5</td>
+                  </tr>
+                  <tr>
+                    <td>2.43</td>
+                    <td>0.48</td>
+                  </tr>
+                  <tr>
+                    <td>2.56</td>
+                    <td>0.46</td>
+                  </tr>
+                  <tr>
+                    <td>2.29</td>
+                    <td>0.4</td>
+                  </tr>
+                  <tr>
+                     <td>2.28</td>
+                    <td>0.4</td>
+                  </tr>
+                 </tbody>
+              </table>
              </div>
-            <br/><br/>
+             <div class="column">
-             <div id="cdwcorrelation" class="section-container">
+              <p>
-                <h2 class="title is-3">CDW correlation of algae <em>Chlorella vulgaris</em> Results</h2>
+                 The cellular dry weight in g/l was then plotted against the
-                <p>
+                optical density measured at 750 nm. The plot with the
-                    By plotting the measured optical densities against the means of the calculated cellular dry weights,
+                 corresponding trend line is shown in the following figure.
-                    a correlation was obtained. It is shown in the following figure.
-                 </p>
-                <br/>
-                <img style="display: block; max-width: 500px; margin: 0 auto;"
-                     src="https://2019.igem.org/wiki/images/a/ad/T--Stuttgart--FigureOD-CDW_correlation_of_algae_Chlorella_vulgaris_Results1.png"/>
-                <small>Figure 1 - OD-CDW correlation of the algae <em>Chlorella vulgaris</em>. Mean of cellular dry
-                    weight in g/L (n=2) was plotted against the measured optical density at 750 nm. Trend line was shown
-                    in red. </small>
-                 <br>
-                <br>
-                <br>
-                <p>The trend line in figure 1 is poorly matching the trend of the measurement points. For this reason,
-                    the correlation curve was rejected. For improvement of this experiment, measurements should be
-                    performed only by one experimenter to reduce pipetting errors or other handling mistakes. Also the
-                    measurements should be taken over a longer time period to gain more trust worthy results.</p>
-                <br>
-            </div>
-            <br/><br/>
-            <div id="cdwodcorrelation" class="section-container">
-                <h2 class="title is-3">CDW-OD correlation by dilution Results</h2>
-                <p>
-                    In the following table you can see the calculated cell dry weights with the corresponding optical
-                    density of the tubes. Some tubes broke during the experiment so corresponding measurements could not
-                    occur.
-                    <br/>
-                    <br/>
-                    <div class="columns">
-                        <div class="column">
-                            <small>Table 2 -Calculated values of the cellular dry weight in g/l with the corresponding
-                                optical
-                                density measured at 750 nm. </small>
-                            <table class="table is-fullwidth">
+                <img
-                                <thead>
+                  style="display: block; max-width: 500px; margin: 0 auto;"
-                                <tr>
+                  src="https://2019.igem.org/wiki/images/d/df/T--Stuttgart--FigureOD-CDW_correlation_of_Chlorella_vulgaris_by_dilution_Results1.png"
-                                    <th>OD</th>
+                />
-                                    <th>CDW[g/l]</th>
+                <small
-                                </tr>
+                  >Figure 1 - Cellular dry weight in g/l is plotted against the
-                                </thead>
+                  optical density measured at 750 nm. The linear fit is shown in
-                                <tbody>
+                  blue together with its formula.</small
-                                <tr>
+                >
-                                    <td>4.87</td>
+                 <br />
-                                    <td>0.98</td>
+                <br />
-                                </tr>
+              </p>
-                                <tr>
-                                    <td>4.41</td>
-                                    <td>0.88</td>
-                                </tr>
-                                <tr>
-                                    <td>4.44</td>
-                                    <td>0.91</td>
-                                </tr>
-                                <tr>
-                                    <td>4.02</td>
-                                    <td>0.8</td>
-                                </tr>
-                                <tr>
-                                    <td>3.92</td>
-                                    <td>0.82</td>
-                                </tr>
-                                <tr>
-                                    <td>3.52</td>
-                                    <td>0.68</td>
-                                </tr>
-                                <tr>
-                                    <td>3.57</td>
-                                    <td>0.71</td>
-                                </tr>
-                                <tr>
-                                    <td>3.03</td>
-                                    <td>0.57</td>
-                                </tr>
-                                <tr>
-                                    <td>2.65</td>
-                                    <td>0.49</td>
-                                </tr>
-                                <tr>
-                                    <td>2.8</td>
-                                    <td>0.5</td>
-                                </tr>
-                                <tr>
-                                    <td>2.43</td>
-                                    <td>0.48</td>
-                                </tr>
-                                <tr>
-                                    <td>2.56</td>
-                                    <td>0.46</td>
-                                </tr>
-                                <tr>
-                                    <td>2.29</td>
-                                    <td>0.4</td>
-                                </tr>
-                                <tr>
-                                    <td>2.28</td>
-                                    <td>0.4</td>
-                                </tr>
-                                </tbody>
-                            </table>
-                        </div>
-                        <div class="column">
-                <p>
-                    The cellular dry weight in g/l was then plotted against the optical density measured at 750 nm. The
-                    plot with the corresponding trend line is shown in the following figure.
-                 </p>
-                <img style="display: block; max-width: 500px; margin: 0 auto;"
+              <p>
-                     src="https://2019.igem.org/wiki/images/d/df/T--Stuttgart--FigureOD-CDW_correlation_of_Chlorella_vulgaris_by_dilution_Results1.png"/>
+                 The slope of the formula further been used for fast estimation
-                 <small>Figure 1 - Cellular dry weight in g/l is plotted against the optical density measured at 750 nm.
+                of the CDW by measuring the optical density at 750 nm.
-                    The linear fit is shown in blue together with its formula.</small>
+              </p>
-                <br>
-                <br>
-                <p>The slope of the formula further been used for fast estimation of the CDW by measuring the optical
-                    density at 750 nm. </p>
              </div>
+          </div>
          </div>
+      </div>
      </div>
-</div>
+  </div>
 </html>

Difference between revisions of "Team:Stuttgart/Results"

Revision as of 21:00, 20 October 2019

Project

Results

qRT-PCR for the relative quantification of specific tRNA-species

References

Cloning of tRNA fragments into pSB1C3

Cloning of tRNA fragments into ptRNA_backbone via BioBrick Cloning

Cloning of tRNA fragments into ptRNA_backbone via Gibson Assembly

Isolation of Vibrio natriegens DSM 759 genome chr.1 and amplification of tRNAs

Cloning of tRNA fragments (amplified from Vibrio natriegens DSM 759 genome) into ptRNA_backbone via NEBuilder HiFi DNA Assembly

Cloning of tRNA fragments (amplified from Vibrio natriegens DSM 759 genome) into ptRNA_backbone via Gibson Assembly

Media based on algae: first tests determining important substrates

Medium based on LB

Medium without tryptone

Autolysis in combination with bead-milling Results

Free amino acid estimation with rFAN assay

Anthrone assay to Determine Soluble Carbohydrate Concentration

References

CDW correlation of algae Chlorella vulgaris Results

CDW-OD correlation by dilution Results