Revision as of 21:06, 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>
+            <li>
-                        <a href="#autolysis"
+              <a href="#autolysis"
-                        >Autolysis in combination <br/>
+                >Autolysis in combination <br />
-                            with bead-milling Results</a
+                with bead-milling Results</a
-                        >
+              >
-                    </li>
+            </li>
-                    <li><a href="#cdwcorrelation">CDW correlation</a></li>
+            <li><a href="#cdwcorrelation">CDW correlation</a></li>
-                    <li>
+            <li>
-                        <a href="#cdwodcorrelation">CDW-OD correlation by dilution Results</a>
+              <a href="#cdwodcorrelation">CDW-OD correlation by dilution Results</a>
-                    </li>
+            </li>
-                </ul>
+          </ul>
-            </aside>
+        </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> 3, 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
+&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 2<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">
+          <div class="columns">
-                    qRT-PCR for the relative quantification of specific tRNA-species
+            <div class="column">
-                </h2>
+              <p>
-                <p>
+                The tRNA fragments were synthesized by IDT and amplified by PCR according to the PCR protocol (<a
-                    Alongside with the generation of a climate-friendly medium, the goal of our project PhyCoVi was to
+                  href="https://2019.igem.org/wiki/images/f/f4/T--Stuttgart--Protocol_PCR.pdf"
-                    optimize
+                  >Protocol_PCR.pdf</a
-                    the strain
+                >). The amplified tRNA fragments were validated via agarose gel electrophoresis (<a
-                    <em>Vibrio natriegens</em> for a potential use in the biotech industry. The optimization is
+                  href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
-                    performed on the
+                  >Stuttgart--Protocol_Agarose_Gel.pdf</a
-                    genomic level to increase the intracellular availability of tRNA species. As a result, the strain&rsquo;s
+                >). Looking at Figure 1 all tRNA fragments showed a clear band at the desired height.
-                    performance to express heterologous proteins is enhanced.
+              </p>
-                </p>
+              <img
-                <br/>
+                style="display: block; max-width: 500px; margin: 0 auto;"
-                <p>
+                src="https://2019.igem.org/wiki/images/thumb/8/86/T--Stuttgart--Amplified_tRNA_fragments.png/592px-T--Stuttgart--Amplified_tRNA_fragments.png"
-                    A method needs to be developed to quantify individual tRNA species specifically to prove the
+              />
-                    increased
+              <small>
-                    expression not only on the protein level. &nbsp;Multiple methods can be found to quantify non-coding
+                Figure 1: Amplified tRNA fragments. The tRNA fragments AGA, CGC, TGC, TCC and AGG were amplified via
-                    RNA
+                PCR. The PCR products were separated by agarose gel electrophoresis. A 2% agarose gel was prepared and
-                    <sup>1, 2</sup> or total tRNA concentration<sup> 3, 4</sup>. Whereas finding a well-established
+&micro;L were loaded for each probe ((1): AGA, (2): CGC, (3): TGC, (4): TCC, (5): AGG). 3 &micro;L of
-                    method to
+                Hyberladder 1 kb Bioline were loaded as a marker (M). The gel was run at 90 V for 1 hour and stained
-                    quantify single tRNA species specifically is in vain. The only method paper was published in the
+                using MidoriGreen.
-                    journal
+              </small>
-                    &ldquo;RNA biology&rdquo; in 2015 by Honda <em>et al.</em>: &ldquo;Four-leaf clover qRT-PCR: A
+              <br />
-                    convenient
+              <br />
-                    method for selective quantification of mature tRNA&rdquo; <sup>5</sup>. The authors of this paper
+              <p>
-                    removed
+                In a first step, the tRNA fragments were cloned into the pSB1C3 vector. The pSB1C3 and the tRNA
-                    the amino acid at the 3&rsquo; end followed by hybridization and ligation with a DNA/RNA hybrid stem
+                fragments AGA, AGG, CGG, TGC, TCC and a combined tRNA fragment containing all 5 tRNAs were digested
-                    loop
+                using the restriction enzymes XbaI and SpeI. <br />
-                    creating a &ldquo;four-leaf clover&rdquo; shaped appearance of the tRNA ligation product. The stem
+                After purification, (<a
-                    loop
+                  href="https://2019.igem.org/wiki/images/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf"
-                    adaptor contained a TaqMan probe binding site. During the qPCR the TaqMan probe was cut by
+                >
-                    exonuclease
+                  Protocol_Clean_and_Concentrate.pdf</a
-                    function of the used polymerase resulting in emission of fluorescence.
+                >) the digested fragments were ligated using the T4 DNA ligase (see
-                </p>
+                <a href="https://2019.igem.org/wiki/images/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf">
-                <br/>
+                  Protocol_BioBrick_Cloning.pdf</a
-                <p>
+                >). The ligated DNA fragments were transformed into DH5&alpha; (<a
-                    Building on the work of Honda <em>et al.</em> we developed a new and simplified method for relative
+                  href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf"
-                    quantification of specific tRNA species without the necessity of TaqMan probes. Instead using a
+                >
-                    DNA/RNA
+                  Protocol_Transformation.pdf</a
-                    hybrid stem loop we used a linear DNA/RNA construct as adaptor.
+                >). The plasmid obtained from the colonies (<a
-                </p>
+                  href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf"
-                <br/>
+                >
-                <p>
+                  Protocol_Plasmid_Preparation.pdf</a
-                    The first step is to isolate RNA with a length of &lt; 200 nt from cultured <em>V. natriegens</em>
+                >) was digested with XbaI to gain linear Plasmid and separated by agarose gel electrophoresis (<a
-                    cells.
+                  href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
-                    Then, the amino acid bound to the 3&rsquo; end needs to be removed by a deacylation reaction. This
+                >
-                    results
+                  Protocol_Agarose_Gel.pdf</a
-                    in a sticky end, where a linear RNA/DNA hybrid adaptor can be ligated, which is complementary to the
+                >). Looking at Figure 2 the obtained plasmids showed no insert, only pSB1C3. Also visible is, that
-&rsquo; end overhang. Although different tRNAs show differences in length and sequence, the last
+                despite the digestion, circular, supercoiled and open circular structures of the plasmid are still
-                    three
+                present. This indicates inefficient digestion by the restriction enzymes.
-                    nucleotides at the 3&rsquo; end are the same for all tRNA species. The ligated adaptor contains a
+              </p>
-                    binding
+              <br />
-                    site for the forward-primer, which is identical for all tRNAs (unspecific primer). We used
+              <img
-                    T4-RNA-ligase 2
+                style="display: block; max-width: 500px; margin: 0 auto;"
-                    that requires ATP. For this reason, a polynucleotide kinase was necessary to carry out a
+                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"
-                    phosphorylation
+              />
-                    reaction at the 5&rsquo; end.
+              <small>
-                </p>
+                Figure 2 - Cloning of tRNA fragments into pSB1C3. The pSB1C3 and the tRNA fragments were digested using
-                <br/>
+                the restriction enzymes XbaI and SpeI. After purification, the digested fragments were ligated using the
-                <p>
+                T4 DNA ligase. The ligated DNA fragments were transformed into DH5&alpha; and subsequently prepared. The
-                    To amplify single tRNA species specifically, we distinguished between two options. First option was
+                obtained plasmids were digested with XbaI before the agarose gel electrophoresis. A 1% agarose gel was
-                    using
+                prepared and 10 &micro;L were loaded for each probe ((1): AGA, (2): AGG, (3): CGG, (4): TGC, (5): TCC,
-                    the specific tRNA primer in a reverse transcription to convert the whole tRNA pool to cDNA.
+                (6): Combined tRNA fragment). As a control (C) the linear pSB1C3 was loaded. 3 &micro;L of GeneRuler,
-                    Following RNase
+kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using
-                    H digestion results in pure cDNA of the desired tRNA species.
+                GelRed.
-                </p>
+              </small>
-                <p>
+              <br />
-                    Later the desired tRNA species is amplified during a qPCR by using the specific reverse primer and
+              <br />
-                    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 2<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 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
-&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,
-kb 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 XbaI and SpeI, it was performed again with
-                            performed again with
+                the enzymes EcoRI-HF and PstI. The pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC, TCC and a combined
-                            the enzymes EcoRI-HF and PstI. The pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC, TCC and
+                tRNA fragment were digested using the restriction enzymes EcoRI-HF and PstI.<br />
-                            a combined
+                After purification, (<a
-                            tRNA fragment were digested using the restriction enzymes EcoRI-HF and PstI.<br/>
+                  href="https://2019.igem.org/wiki/images/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf"
-                            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
-                            Protocol_Clean_and_Concentrate.pdf</a
+                <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 (see
+                  Protocol_BioBrick_Cloning.pdf</a
-                            <a href="https://2019.igem.org/wiki/images/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf">
+                >). The ligated DNA fragments were transformed into DH5&alpha; cells (<a
-                                Protocol_BioBrick_Cloning.pdf</a
+                  href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf"
-                            >). The ligated DNA fragments were transformed into 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
-                            Protocol_Transformation.pdf</a
+                  href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf"
-                        >). 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
-                            Protocol_Plasmid_Preparation.pdf</a
+                  href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
-                        >) was digested with EcoRI-HF to gain linear Plasmid and separated by agarose gel
+                  >Protocol_Agarose_Gel.pdf</a
-                            electrophoresis (<a
+                >). The agarose gel revealed no successful cloning as all obtained plasmids showed no insert, only
-                                href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
+                pSB1C3 (data not shown).
-                        >Protocol_Agarose_Gel.pdf</a
+              </p>
-                        >). The agarose gel revealed no successful cloning as all obtained plasmids showed no insert,
+              <br />
-                            only
-                            pSB1C3 (data not shown).
-                        </p>
-                        <br/>
-                    </div>
-                    <div class="column">
-                        <h2 class="title is-4">
-                            Cloning of tRNA fragments into ptRNA_backbone via BioBrick Cloning
-                        </h2>
-                        <p>
+              <h2 class="title is-4">
-                            In a first step, the self-designed linear ptRNA_backbone from IDT was ligated according to
+                Cloning of tRNA fragments into ptRNA_backbone via BioBrick Cloning
-                            the NEB
+              </h2>
-                            Ligation Protocol with T4 DNA Ligase (see
-                            <a href="https://2019.igem.org/wiki/images/2/2e/T--Stuttgart--Blunt_End_Ligation.pdf">
-                                T--Stuttgart--Blunt_End_Ligation.pdf</a
-                            >). 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">
-                                Protocol_Transformation.pdf</a
-                            >). Successfully transformed DH5&alpha; 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 (<a
-                                href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf"
-                        >
-                            Protocol_Plasmid_Preparation.pdf</a
-                        >). The plasmid obtained from the colonies 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
-                        >). Looking at Figure 3 all plasmids run at the desired length which corresponds to the length
-                            of the
-                            ptRNA_backbone 2159 bp.
-                        </p>
-                        <br/>
-                        <br/>
-                        <img
-                                style="display: block; max-width: 500px; margin: 0 auto;"
-                                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"
-                        />
-                        <small style="text-align: justify">
-                            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 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): ptRNA_backbone gBlock from
-                            IDT, (2):
-                            colony 2, (3): colony 3, (4): colony 4, (5): colony 5, (6): colony 6). 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>
-                            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, (<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 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 4 reveals no successful cloning as all obtained Plasmids show no insert, only
-                            ptRNA_backbone.
-                        </p>
-                        <img
+              <p>
-                                style="display: block; max-width: 500px; margin: 0 auto;"
+                In a first step, the self-designed linear ptRNA_backbone from IDT was ligated according to the NEB
-                                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"
+                Ligation Protocol with T4 DNA Ligase (see
-                        />
+                <a href="https://2019.igem.org/wiki/images/2/2e/T--Stuttgart--Blunt_End_Ligation.pdf">
-                        <small>
+                  T--Stuttgart--Blunt_End_Ligation.pdf</a
-                            Figure 4 - Cloning of tRNA fragments into ptRNA_backbone. The ptRNA_backbone and the tRNA
+                >). Afterward, the ligated ptRNA-backbone was transformed in <em>E. coli</em> DH5a cells (see
-                            fragments were
+                <a href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf">
-                            digested using the restriction enzymes EcoRI-HF and PstI. After purification, the digested
+                  Protocol_Transformation.pdf</a
-                            fragments
+                >). Successfully transformed DH5&alpha; cells were selected on LB agar plates containing tetracycline.
-                            were ligated using the T4 DNA ligase. The ligated DNA fragments were transformed into DH5&alpha;
+                The next day the circular ptRNA_backbone was prepared from the colonies according to the Plasmid
-                            and
+                Preparation protocol (<a
-                            subsequently prepared. The obtained plasmids were digested with EcoRI-HF before the agarose
+                  href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf"
-                            gel
+                >
-                            electrophoresis. A 1% agarose gel was prepared and 10 &micro;L were loaded for each probe
+                  Protocol_Plasmid_Preparation.pdf</a
-                            ((1): AGA,
+                >). The plasmid obtained from the colonies was digested with EcoRI-HF to gain linear Plasmid and
-                            (2): AGG, (3): CGG, (4): TGC, (5): TCC, (6): Combined tRNA fragment). As a control (C) the
+                separated by agarose gel electrophoresis (<a
-                            linear
+                  href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
-                            ptRNA_backbone was loaded. 3 &micro;L of GeneRuler, 1kb Plus DNA Ladder was loaded as a
+                >
-                            marker (M). The
+                  Protocol_Agarose_Gel.pdf</a
-                            gel was run at 90 V for 1 hour and stained using GelRed.
+                >). Looking at Figure 3 all plasmids run at the desired length which corresponds to the length of the
-                        </small>
+                ptRNA_backbone 2159 bp.
-                        <br/>
+              </p>
-                        <p>
+              <br />
-                            This BioBrick cloning was repeated several times using different EcoRI-HF and PstI stocks.
+              <br />
-                            Following
+              <img
-                            transformation in DH5&alpha; revealed no successful cloning. The colonies obtained showed no
+                style="display: block; max-width: 500px; margin: 0 auto;"
-                            insert in
+                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"
-                            an agarose gel and only ptRNA_backbone. Following transformation in competent Vibrio
+              />
-                            natriegens cells
+              <small style="text-align: justify">
-                            also revealed no successful cloning (data not shown).
+                Figure 3 -Cloning of ptRNA_backbone. The linear ptRNA_backbone fragment from IDT was ligated using the
-                        </p>
+                T4 DNA Ligase. The ligated ptRNA-backbone was transformed into DH5&alpha; and subsequently prepared. The
-                        <br/>
+                obtained plasmids were digested with EcoRI-HF before the agarose gel electrophoresis. A 1% agarose gel
-                        <br/>
+                was prepared and 10 &micro;L were loaded for each probe ((1): ptRNA_backbone gBlock from IDT, (2):
-                        <h2 class="title is-4">
+                colony 2, (3): colony 3, (4): colony 4, (5): colony 5, (6): colony 6). 3 &micro;L of GeneRuler, 1kb Plus
-                            Cloning of tRNA fragments into ptRNA_backbone via Gibson Assembly
+                DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using GelRed.
-                        </h2>
+              </small>
+              <br />
+              <br />
+              <p>
+                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, (<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 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 4 reveals no successful cloning as all obtained Plasmids show no insert, only ptRNA_backbone.
+              </p>
-                        <p>
+              <img
-                            Cloning of tRNA fragments into ptRNA_backbone was also performed using Gibson Assembly.
+                style="display: block; max-width: 500px; margin: 0 auto;"
-                            Gibson Assembly
+                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"
-                            was conducted according to the protocol Gibson Assembly (<a
+              />
-                                href="https://2019.igem.org/wiki/images/6/6c/T--Stuttgart--Protocol_Gibson_Assembly.pdf"
+              <small>
-                        >
+                Figure 4 - Cloning of tRNA fragments into ptRNA_backbone. The ptRNA_backbone and the tRNA fragments were
-                            Protocol_Gibson_Assembly.pdf</a
+                digested using the restriction enzymes EcoRI-HF and PstI. After purification, the digested fragments
-                        >). The Gibson reaction was transformed into competent DH5&alpha; cells (<a
+                were ligated using the T4 DNA ligase. The ligated DNA fragments were transformed into DH5&alpha; and
-                                href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf"
+                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,
-                            Protocol_Transformation.pdf</a
+                (2): AGG, (3): CGG, (4): TGC, (5): TCC, (6): Combined tRNA fragment). As a control (C) the linear
-                        >). The plasmid obtained from the colonies (<a
+                ptRNA_backbone was loaded. 3 &micro;L of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The
-                                href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf"
+                gel was run at 90 V for 1 hour and stained using GelRed.
-                        >
+              </small>
-                            Protocol_Plasmid_Preparation.pdf</a
+              <br />
-                        >) was digested with EcoRI-HF to gain linear Plasmid and separated by agarose gel
+              <p>
-                            electrophoresis (<a
+                This BioBrick cloning was repeated several times using different EcoRI-HF and PstI stocks. Following
-                                href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
+                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
-                            Protocol_Agarose_Gel.pdf</a
+                also revealed no successful cloning (data not shown).
-                        >). Figure 5 reveals no successful cloning as all obtained Plasmids show no insert, only
+              </p>
-                            ptRNA_backbone.
+              <br />
-                        </p>
+              <br />
-                        <img
+              <h2 class="title is-4">
-                                style="display: block; max-width: 500px; margin: 0 auto;"
+                Cloning of tRNA fragments into ptRNA_backbone via Gibson Assembly
-                                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"
+              </h2>
-                        />
-                        <small
+              <p>
-                        >Figure 5 -Gibson Assembly of tRNA fragments into ptRNA_backbone. Gibson Assembly was performed
+                Cloning of tRNA fragments into ptRNA_backbone was also performed using Gibson Assembly. Gibson Assembly
-                            according to the Gibson Assembly Protocol. The Gibson Assembly reaction was transformed into
+                was conducted according to the protocol Gibson Assembly (<a
-                            DH5&alpha;
+                  href="https://2019.igem.org/wiki/images/6/6c/T--Stuttgart--Protocol_Gibson_Assembly.pdf"
-                            and subsequently prepared. The obtained plasmids were digested with EcoRI-HF before the
+                >
-                            agarose gel
+                  Protocol_Gibson_Assembly.pdf</a
-                            electrophoresis. A 1% agarose gel was prepared and 10 &micro;L were loaded for each probe
+                >). The Gibson reaction was transformed into competent DH5&alpha; cells (<a
-                            ((1): AGA,
+                  href="https://2019.igem.org/wiki/images/8/83/T--Stuttgart--Protocol_Transformation.pdf"
-                            (2): AGG, (3): CGG, (4): TGC, (5): TCC). As a control (C) the linear ptRNA_backbone was
+                >
-                            loaded. 3
+                  Protocol_Transformation.pdf</a
-                            &micro;L of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90
+                >). The plasmid obtained from the colonies (<a
-                            V for 1
+                  href="https://2019.igem.org/wiki/images/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf"
-                            hour and stained using GelRed.</small
+                >
-                        >
+                  Protocol_Plasmid_Preparation.pdf</a
-                        <br/>
+                >) was digested with EcoRI-HF to gain linear Plasmid and separated by agarose gel electrophoresis (<a
-                        <br/>
+                  href="https://2019.igem.org/wiki/images/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf"
-                        <p>
+                >
-                            Cloning of tRNA fragments into ptRNA_backbone via Gibson Assembly was repeated with another
+                  Protocol_Agarose_Gel.pdf</a
-                            Gibson
+                >). Figure 5 reveals no successful cloning as all obtained Plasmids show no insert, only ptRNA_backbone.
-                            Assembly Master Mix and revealed no successful cloning. The colonies obtained showed no
+              </p>
-                            insert in an
+              <img
-                            agarose gel and only ptRNA_backbone (data not shown).
+                style="display: block; max-width: 500px; margin: 0 auto;"
-                        </p>
+                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"
-                        <br/>
+              />
-                        <br/>
+              <small
-                        <h2 class="title is-4">
+                >Figure 5 -Gibson Assembly of tRNA fragments into ptRNA_backbone. Gibson Assembly was performed
-                            Isolation of <em>Vibrio natriegens</em> DSM 759 genome chr.1 and amplification of tRNAs
+                according to the Gibson Assembly Protocol. The Gibson Assembly reaction was transformed into DH5&alpha;
-                        </h2>
+                and subsequently prepared. The obtained plasmids were digested with EcoRI-HF before the agarose gel
-                        <p>
+                electrophoresis. A 1% agarose gel was prepared and 10 &micro;L were loaded for each probe ((1): AGA,
-                            As an alternative to cloning of tRNA fragments provided by IDT, the tRNAs AGA, AGG, CGG, TGC
+                (2): AGG, (3): CGG, (4): TGC, (5): TCC). As a control (C) the linear ptRNA_backbone was loaded. 3
-                            and TCC
+                &micro;L of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1
-                            were amplified from the
+                hour and stained using GelRed.</small
-                            <em>Vibrio natriegens</em> DSM 759 genome. Therefore, the <em>Vibrio natriegens</em> DSM 759
+              >
-                            genome
+              <br />
-                            chr.1 was isolated according to the gDNA Extraction protocol (<a
+              <br />
-                                href="https://2019.igem.org/wiki/images/c/c0/T--Stuttgart--gDNA_extraction.pdf"
+              <p>
-                        >
+                Cloning of tRNA fragments into ptRNA_backbone via Gibson Assembly was repeated with another Gibson
-                            gDNA_extraction.pdf</a
+                Assembly Master Mix and revealed no successful cloning. The colonies obtained showed no insert in an
-                        >). The tRNA fragments AGA, AGG, CGG, TGC, TCC were amplified from the <em>Vibrio
+                agarose gel and only ptRNA_backbone (data not shown).
-                            natriegens</em> DSM
+              </p>
-genome via PCR with appropriate primers (see
+              <br />
-                            <a href="https://2019.igem.org/wiki/images/f/f4/T--Stuttgart--Protocol_PCR.pdf">
+              <br />
-                                Protocol_PCR.pdf</a>).
+              <h2 class="title is-4">
-                            Looking at Figure 6 all tRNA fragments showed a clear band at the desired height. The tRNA
+                Isolation of <em>Vibrio natriegens</em> DSM 759 genome chr.1 and amplification of tRNAs
-                            fragments
+              </h2>
-                            were extracted from the agarose gel according to the gel extraction protocol (<a
+              <p>
-                                href="https://2019.igem.org/wiki/images/3/32/T--Stuttgart--Protocol_Gel_Extraction.pdf"
+                As an alternative to cloning of tRNA fragments provided by IDT, the tRNAs AGA, AGG, CGG, TGC and TCC
-                        >
+                were amplified from the
-                            Protocol_Gel_Extraction.pdf</a
+                <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
-                        </p>
+                  href="https://2019.igem.org/wiki/images/c/c0/T--Stuttgart--gDNA_extraction.pdf"
-                        <br/>
+                >
-                        <br/>
+                  gDNA_extraction.pdf</a
-                        <img
+                >). The tRNA fragments AGA, AGG, CGG, TGC, TCC were amplified from the <em>Vibrio natriegens</em> DSM
-                                style="display: block; max-width: 500px; margin: 0 auto;"
+genome via PCR with appropriate primers (see
-                                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"
+                <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
-                        <small
+                were extracted from the agarose gel according to the gel extraction protocol (<a
-                        >Figure 6: Amplification of tRNAs from the Vibrio natriegens DSM 759 genome. The Vibrio
+                  href="https://2019.igem.org/wiki/images/3/32/T--Stuttgart--Protocol_Gel_Extraction.pdf"
-                            natriegens DSM
+                >
-genome chr.1 was isolated according to the Protocol gDNA Extraction. The tRNA fragments
+                  Protocol_Gel_Extraction.pdf</a
-                            AGA, AGG,
+                >).
-                            CGG, TGC, TCC were amplified from the Vibrio natriegens DSM 759 genome via PCR with
+              </p>
-                            appropriate primers.
+              <br />
-                            A 1.5 % agarose gel was prepared and 10 &micro;L were loaded for each probe ((1): AGA, (2):
+              <br />
-                            AGG, (3):
+              <img
-                            CGG, (4): TGC, (5): TCC). 3 &micro;L of GeneRuler, 1kb Plus DNA Ladder was loaded as a
+                style="display: block; max-width: 500px; margin: 0 auto;"
-                            marker (M). The
+                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"
-                            gel was run at 90 V for 1 hour and stained using GelRed.</small
+              />
-                        >
+              <small
-                        <br/>
+                >Figure 6: Amplification of tRNAs from the Vibrio natriegens DSM 759 genome. The Vibrio natriegens DSM
-                     </div>
+genome chr.1 was isolated according to the Protocol gDNA Extraction. The tRNA fragments AGA, AGG,
-                 </div>
+                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 />
+                <h2 class="title is-4">
+                     Cloning of tRNA fragments (amplified from <em>Vibrio natriegens</em> DSM 759 genome) into ptRNA_backbone via
+                    BioBrick Cloning
+                 </h2>
-                <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
+                     <em>Vibrio natriegens</em> DSM 759 genome were digested using the restriction enzymes EcoRI-HF and PstI.
-                    PstI.
                      After purification, (<a
                          href="https://2019.igem.org/wiki/images/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf"
@@ Line 614: / Line 497: @@
                  >
                      Protocol_Plasmid_Preparation.pdf</a
-                 >) was digested with EcoRI-HF and PstI to release inserted tRNA fragments from the ptRNA_backbone and
+                 >) was digested with EcoRI-HF and PstI to release inserted tRNA fragments from the ptRNA_backbone and gain
-                    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"
@@ Line 629: / Line 511: @@
                  <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
+                     amplified from the Vibrio natriegens DSM 759 genome. The ptRNA_backbone and the tRNA fragments were digested
-                    digested
+                     using the restriction enzymes EcoRI-HF and PstI. After purification, the digested fragments were ligated
-                     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
+                     prepared. The obtained plasmids were digested with EcoRI-HF and PstI before the agarose gel electrophoresis.
-                    electrophoresis.
+                     A 1% agarose gel was prepared and 10 &micro;L were loaded for each probe ((1): AGA, (2): AGG, (3): CGG, (4):
-                     A 1% agarose gel was prepared and 10 &micro;L were loaded for each probe ((1): AGA, (2): AGG, (3):
+                     TGC, (5): ACC). As a control (C) the linear ptRNA_backbone was loaded. 3 &micro;L of GeneRuler, 1kb Plus DNA
-                    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
+                     Following transformation in competent Vibrio natriegens cells also revealed no successful cloning (data not
-                    (data not
                      shown).
                  </p>
-                 <br/>
+                 <br />
-                 <br/>
+                 <br />
                  <h3 class="title is-4">
                      Cloning of tRNA fragments (amplified from
@@ Line 655: / Line 531: @@
                  <p>
                      The tRNA fragments were previously amplified from
-                     <em>Vibrio natriegens</em> DSM 759 genome. The NEBuilder HiFi DNA Assembly was performed according
+                     <em>Vibrio natriegens</em> DSM 759 genome. The NEBuilder HiFi DNA Assembly was performed according to the
-                    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
+                 >). Following transformation into competent DH5&alpha; cells revealed no successful cloning as no colonies
-                    colonies
+                     were obtained. Following transformation in competent <em>Vibrio natriegens</em> cells also revealed no
-                     were obtained. Following transformation in competent <em>Vibrio natriegens</em> cells also revealed
-                    no
                      successful cloning (data not shown).
                  </p>
-                 <br/>
+                 <br />
-                 <br/>
+                 <br />
                  <h3 class="title is-4">
                      Cloning of tRNA fragments (amplified from
@@ Line 679: / Line 552: @@
                  >
                      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
+                 >). Due to the orientation in the <em>Vibrio natriegens</em> DSM 759 genome only the tRNA fragments AGG and
-                    and
+                     TGC could be used for Gibson Assembly. The Gibson reaction was transformed into competent DH5&alpha; cells
-                     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
@@ Line 689: / Line 560: @@
                  >
                      Protocol_Plasmid_Preparation.pdf</a
-                 >) was digested with EcoRI-HF and PstI to release inserted tRNA fragments from the ptRNA_backbone and
+                 >) was digested with EcoRI-HF and PstI to release inserted tRNA fragments from the ptRNA_backbone and gain
-                    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"
@@ Line 703: / Line 573: @@
                  />
                  <small>
-                     Figure 8: Gibson Assembly of tRNA fragments into ptRNA_backbone. Gibson Assembly was performed
+                     Figure 8: Gibson Assembly of tRNA fragments into ptRNA_backbone. Gibson Assembly was performed according to
-                    according to
+                     the Gibson Assembly Protocol. The tRNA fragments were previously amplified from the Vibrio natriegens DSM
-                     the Gibson Assembly Protocol. The tRNA fragments were previously amplified from the Vibrio
+genome. The Gibson Assembly reaction was transformed into DH5&alpha; and subsequently prepared. The
-                    natriegens DSM
+                     obtained plasmids were digested with EcoRI-HF and PstI before the agarose gel electrophoresis. A 1% agarose
-genome. The Gibson Assembly reaction was transformed into DH5&alpha; and subsequently prepared.
+                     gel was prepared and 10 &micro;L were loaded for each probe ((1): AGG colony 1, (2): AGG colony 2, (3): TGC
-                    The
+                     colony 1, (4): TGC colony 2). 3 &micro;L of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). 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/>
+                 <br />
                  <p>
-                     Cloning of tRNA fragments into ptRNA_backbone via Gibson Assembly was repeated with another Gibson
+                     Cloning of tRNA fragments into ptRNA_backbone via Gibson Assembly was repeated with another Gibson Assembly
-                    Assembly
+                     Master Mix and revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and
-                     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>
-            <div id="algae-media-based" class="section-container">
+        </div>
-                <h2 class="title is-3">
+        <br /><br />
-                    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>
-                <div class="has-text-centered" style="width: 75%; margin:0 auto;">
+        <div id="algae-media-based" class="section-container">
-                    <canvas id="autolysis-figure-1"></canvas>
+          <h2 class="title is-3">
-                </div>
+            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: [
-                                    {
-                                        backgroundColor: ['#3e95cd', '#3e95cd', '#3e95cd', '#3e95cd', '#3e95cd', '#3e95cd'],
-                                        label: 'Population (millions)',
-                                        data: [3.132222386, 4.852523798, 0.021842627, 0.037566219, 0.043678259, 0.087077261]
-                                    }
-                                ]
-                            },
-                            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
+            window.onload = function() {
-                    of
+              var autolysisfigure1 = document.getElementById('autolysis-figure-1').getContext('2d');
-                    free amino acids [%] relates to the biomass used in the experiment.</small
+              window.myBar = new Chart(autolysisfigure1, {
-                 >
+                 type: 'bar',
-                 <br/>
+                 data: {
-                <p>
+                  labels: [
-                     The highest amounts of free amino acids were reached with yeast at pH 12 with 4.85&nbsp;%. Both
+                     'yeast pH3',
-                     algae showed
+                     'yeast pH12',
-                     very low yield in free amino acids. The best results showed <em>C. sorokiniana</em> at pH&nbsp;12.
+                     'C.vulgaris pH3',
-                     It is
+                     'C.vulgaris pH12',
-                     possible, that the amount of glass beads and the size of the glass beads were to little, which led
+                     'C.sorokeniana pH3',
-                     to less
+                     'C.sorokeniana pH12'
-                    cell wall disruption. Therefore, amino acids would have been retained within the cells. This would
+                  ],
-                     explain
+                  datasets: [
-                    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
+                      backgroundColor: ['#3e95cd', '#3e95cd', '#3e95cd', '#3e95cd', '#3e95cd', '#3e95cd'],
-                    break,
+                      label: 'Population (millions)',
-                    than a plasma membrane. This could explain the difference between the yeast samples and the algae
+                      data: [3.132222386, 4.852523798, 0.021842627, 0.037566219, 0.043678259, 0.087077261]
-                    samples.
+                     }
-                     Due to the low yield in free amino acids, it was decided to investigate other methods for cell
+                  ]
-                    extraction of
+                 },
-                    algae.
+                 options: {
-                 </p>
+                  responsive: true,
-                 <br/>
+                  legend: {
-                <br/>
+                     position: 'top'
-                <h3 class="title is-4">
+                  },
-                     Anthrone assay to Determine Soluble Carbohydrate Concentration
+                  scales: {
-                </h3>
+                     yAxes: [
-                <p>
+                      {
-                     Similar to the rFAN assay the anthrone assay is a method to detect free monosaccharides in a liquid.
+                        scaleLabel: {
-                    Therefor samples from the experiment
+                          display: true,
-                    <a href="https://2019.igem.org/wiki/images/f/f3/T--Stuttgart--Experiments_AnthroneAssay.pdf"
+                          labelString: 'percentage of free amio acids [%]'
-                    >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
+              });
-, right side).
+            };
-                 </p>
+          </script>
-                <img
+          <small
-                        style="display: block; margin:0 auto;"
+            >Figure 1 -Autolysis and subsequent bead-milling of algae C. vulgaris and C. sorokiniana. The percentage of
-                        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"
+            free amino acids [%] relates to the biomass used in the experiment.</small
-                />
+          >
-                <small
+          <br />
-                >Figure 2: Pictures of the anthrone calibration curve as well as the anthrone assay of samples. For the
+          <p>
-                    calibration curve known amounts of glucose is dissolved in water and the optical density at 620 nm
+            The highest amounts of free amino acids were reached with yeast at pH 12 with 4.85&nbsp;%. Both algae showed
-                    is
+            very low yield in free amino acids. The best results showed <em>C. sorokiniana</em> at pH&nbsp;12. It is
-                    measured (left side). This can be used to determine the monosaccharide concentration of anthone
+            possible, that the amount of glass beads and the size of the glass beads were to little, which led to less
-                    treated
+            cell wall disruption. Therefore, amino acids would have been retained within the cells. This would explain
-                    samples which previously underwent autolysis (pH3 or pH6) with or without subsequent bead-mill
+            the little amounts of free amino acids achieved with this method. Also, <em>C. vulgaris </em>and
-                    treatment
+            <em>C. sorokinia </em>have a cell wall, in contrast to yeast <sup>3</sup>>. Cell walls are harder to break,
-                    (RKM) (right side).</small
+            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
+, right side).
+          </p>
-                <br/>
+          <img
-                <br/>
+            style="display: block; margin:0 auto;"
-                <p>
+            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"
-                    One can tell from the coloring of the samples in figure 2, that the carbohydrate concentration
+          />
-                    should differ
+          <small
-                    very slightly between the samples pH3, pH6, bead mill extraction +pH3 and bead mill extraction +pH6.
+            >Figure 2: Pictures of the anthrone calibration curve as well as the anthrone assay of samples. For the
-                    Due to
+            calibration curve known amounts of glucose is dissolved in water and the optical density at 620 nm is
-                    the cloudiness of the control sample, a background corrected optical density could not be
+            measured (left side). This can be used to determine the monosaccharide concentration of anthone treated
-                    determined.
+            samples which previously underwent autolysis (pH3 or pH6) with or without subsequent bead-mill treatment
-                    Therefore, the coloring scheme served as evaluation for successful carbohydrate determination.
+            (RKM) (right side).</small
-                </p>
+          >
-                <p>
-                    Hereby, bead-mill (RKM) with subsequent autolysis at pH3 was determined to be the method of choice.
+          <br />
-                </p>
+          <br />
-                <br/><br/>
+          <p>
-                <div class="notification">
+            One can tell from the coloring of the samples in figure 2, that the carbohydrate concentration should differ
-                    <h3 class="title is-5">References</h3>
+            very slightly between the samples pH3, pH6, bead mill extraction +pH3 and bead mill extraction +pH6. Due to
-                    <ol>
+            the cloudiness of the control sample, a background corrected optical density could not be determined.
-                        <li>
+            Therefore, the coloring scheme served as evaluation for successful carbohydrate determination.
-                            Kim et al., &ldquo;Preparation of flavor-enhancing yeast extract using a Saccharomyces
+          </p>
-                            cerevisiae strain
+          <p>
-                            with high RNA content&rdquo;, Korean J Food Sci Technol, 31 (2) (1999), pp. 475-481.
+            Hereby, bead-mill (RKM) with subsequent autolysis at pH3 was determined to be the method of choice.
-                        </li>
+          </p>
-                        <li>
+          <br /><br />
-                            T.L. Babayan, M.G. Bezrukov, &ldquo;Autolysis in yeasts&rdquo;, Acta Biotechnol, 5 (2)
+          <div class="notification">
-                            (1985), pp.
+            <h3 class="title is-5">References</h3>
--136.
+            <ol>
-                        </li>
+              <li>
-                        <li>
+                Kim et al., &ldquo;Preparation of flavor-enhancing yeast extract using a Saccharomyces cerevisiae strain
-                            van der Rest, M E et al. &ldquo;The plasma membrane of Saccharomyces cerevisiae: structure,
+                with high RNA content&rdquo;, Korean J Food Sci Technol, 31 (2) (1999), pp. 475-481.
-                            function,
+              </li>
-                            and biogenesis.&rdquo; Microbiological reviews vol. 59,2 (1995): 304-22.
+              <li>
-                        </li>
+                T.L. Babayan, M.G. Bezrukov, &ldquo;Autolysis in yeasts&rdquo;, Acta Biotechnol, 5 (2) (1985), pp.
-                        <li>
+-136.
-                            Takeda, &ldquo;Classification of Chlorella strains by cell wall sugar composition&rdquo;
+              </li>
-                            Phytochemistry,
+              <li>
-                            vol. 27, 12, (1988), pp. 3823-3826.
+                van der Rest, M E et al. &ldquo;The plasma membrane of Saccharomyces cerevisiae: structure, function,
-                        </li>
+                and biogenesis.&rdquo; Microbiological reviews vol. 59,2 (1995): 304-22.
-                        <li>
+              </li>
-                            [4} Takeda, &ldquo;Classification of Chlorella strains by cell wall sugar composition&rdquo;
+              <li>
-                            Phytochemistry, vol. 27, 12, (1988), pp. 3823-3826.
+                Takeda, &ldquo;Classification of Chlorella strains by cell wall sugar composition&rdquo; Phytochemistry,
-                        </li>
+                vol. 27, 12, (1988), pp. 3823-3826.
-                    </ol>
+              </li>
-                </div>
+              <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>
+              <table class="table is-fullwidth">
+                <thead>
+                  <tr>
+                    <th>OD</th>
+                    <th>CDW[g/l]</th>
+                  </tr>
+                </thead>
+                <tbody>
+                  <tr>
+                    <td>4.87</td>
+                    <td>0.98</td>
+                  </tr>
+                  <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>
-            <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 optical density measured at 750 nm. The plot
-                 <p>
+                with the corresponding trend line is shown in the following figure.
-                    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;"
+                  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"
+                  src="https://2019.igem.org/wiki/images/d/df/T--Stuttgart--FigureOD-CDW_correlation_of_Chlorella_vulgaris_by_dilution_Results1.png"
                  />
                  <small
-                >Figure 1 - OD-CDW correlation of the algae <em>Chlorella vulgaris</em>. Mean of cellular dry weight in
+                  >Figure 1 - Cellular dry weight in g/l is plotted against the optical density measured at 750 nm. The
-                    g/L
+                  linear fit is shown in blue together with its formula.</small
-                    (n=2) was plotted against the measured optical density at 750 nm. Trend line was shown in red.
+                >
-                </small>
+                <br />
-                <br/>
+                <br />
-                <br/>
+              </p>
-                <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>
-                        <table class="table is-fullwidth">
-                            <thead>
-                            <tr>
-                                <th>OD</th>
-                                <th>CDW[g/l]</th>
-                            </tr>
-                            </thead>
-                            <tbody>
-                            <tr>
-                                <td>4.87</td>
-                                <td>0.98</td>
-                            </tr>
-                            <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.
-                            <img
-                                    style="display: block; max-width: 500px; margin: 0 auto;"
-                                    src="https://2019.igem.org/wiki/images/d/df/T--Stuttgart--FigureOD-CDW_correlation_of_Chlorella_vulgaris_by_dilution_Results1.png"
-                            />
-                            <small
-                            >Figure 1 - Cellular dry weight in g/l is plotted against the optical density measured at
-nm. The
-                                linear fit is shown in blue together with its formula.</small
-                            >
-                            <br/>
-                            <br/>
-                        </p>
-                        <p>
+              <p>
-                            The slope of the formula further been used for fast estimation of the CDW by measuring the
+                The slope of the formula further been used for fast estimation of the CDW by measuring the optical
-                            optical
+                density at 750 nm.
-                            density at 750 nm.
+              </p>
-                        </p>
-                    </div>
-                </div>
              </div>
+          </div>
          </div>
+      </div>
      </div>
-</div>
+  </div>
 </html>

Difference between revisions of "Team:Stuttgart/Results"

Revision as of 21:06, 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 BioBrick Cloning

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