Team:Stuttgart/Results

Project

Results

Vibrio

• qPCR
• CDW correlation
• CDW correlation
• CDW-OD correlation by dilution Results

Vibrio - Wet lab

Week	Experiment	Participents	Description	Results
Week	Experiment	Participents	Description	Results
Week 23 06/03/2019-06/09/2019	ptRNA_backbone -Ligation	Benedikt Schober	The linearized fragment from IDT was ligated using the T4-Ligase. Afterwards the ligated ptRNA-backbone was transformed in E. coli Dh5a cells.	https://2019.igem.org/wiki/images/a/af/T--Stuttgart--ptRNA_backbone.png
Week 24 06/10/2019-06/16/2019	Amplifaction of tRNA-Fragments	Kai Schülke ,Jan Müller	The tRNA fragments were synthetized by IDT and amplified by PCR.	The PCR was sucessful. The DNA was checked and purified via agarose gel electrophoresis.
Week 25 06/17/2019-06/23/2019	competent cells Vibrio natriegens DSM759	Benedikt Schober, Katharina Kocher	competent Vibrio natriegens DSM 759 were made according to iGEM Marburg 2018	Competent vibrio natriegens DSM 759 cells were checked on performance via transformation of the sfGFP-Vector.
Week 26 06/24/2019-06/30/2019	competent cells Escherichia coli DH5a	Kai Schülke, Benedikt Schober, Katharina Kocher	chemically and electro-competent Escherichia coli DH5a cells were made.	Competent escherichia coli cells were checked on performance via transformation of a pUC19a Vector.
Week 27 07/01/2019-07/07/2019	growth curve V.natriegens in BHIN	Jan Müller, Jan Seeger, Katharina Kocher	Growth curves of the new Vibrio natriegens DSM 759 in BHIN without plasmid.	The resulting growth curve is shown below.
Week 28 07/08/2019-07/14/2019	Cloning of tRNA fragments into ptRNA_backbone	Katharina Kocher, Benedikt Schober	The ptRNA_backbone and the tRNA fragments AGA, AGG, CGG, TGC, TCC and the combined tRNA fragment were digested using the restriction enzymes XbaI and SpeI. After purification the digested fragments were ligated using the T4 DNA ligase.	Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA_backbone.
Week 29 07/15/2019-07/21/2019	Cloning of tRNA fragments into ptRNA_backbone	Katharina Kocher, Benedikt Schober	The ptRNA_backbone and the tRNA fragments AGA, AGG, CGG, TGC, TCC and the combined tRNA fragment were digested using the restriction enzymes PstI and EcoRI. After purification the digested fragments were ligated using the T4 DNA ligase.	Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA_backbone.
Week 30 07/22/2019-07/28/2019	Plasmid-Preperation, Agarose-Gel, Agarose-Gel-Extraction, Clean and Concentrator Kit	Katharina Kocher, Benedikt Schober	The ptRNA_backbone and the tRNA fragments AGA, AGG, CGG, TGC, TCC and the combined tRNA fragment were digested using the restriction enzymes PstI and EcoRI. After purification the digested fragments were ligated using the T4 DNA ligase.	Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA_backbone.
Week 31 07/29/2019-08/04/2019	Cloning of tRNA fragements into pSB1C3	Katharina Kocher; Benedikt Schober	The vector pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC, TCC and the combined tRNA fragment were digested using the restriction enzymes XbaI and SpeI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning.	Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only pSB1C3.
Week 32 08/05/2019-08/11/2019	Cloning of tRNA fragments into pSB1C3	Katharina Kocher; Benedikt Schober	The vector pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC, TCC and the combined tRNA fragment were digested using the restriction enzymes XbaI and SpeI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning.	Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only pSB1C3.
Week 33 08/12/2019-08/18/2019	Cloning of tRNA fragments into pSB1C3	Katharina Kocher; Benedikt Schober	The vector pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC, TCC and the combined tRNA fragment were digested using the restriction enzymes EcoRI and PstI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning.	Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only pSB1C3.
Week 34 08/19/2019-08/25/2019	Cloning of tRNA fragments into pSB1C3	Katharina Kocher; Benedikt Schober	The vector pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC, TCC and the combined tRNA fragment were digested using the restriction enzymes EcoRI and PstI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning.	Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only pSB1C3.
Week 35 08/26/2019-09/01/2019	Cloning of tRNA fragments into pSB1C3	Katharina Kocher; Benedikt Schober	After a re-synthesis of the ptRNA_backbone, the tRNA fragments AGA, AGG, CGG, TGC, TCC,the combined tRNA fragment and the pTRNA_backbone were digested using the restriction enzymes EcoRI and PstI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning.	Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA_backbone.
Week 36 09/02/2019-09/08/2019	Cloning of tRNA fragments into ptRNA_backbone	Katharina Kocher; Benedikt Schober	After a re-synthesis of the ptRNA_backbone, the tRNA fragments AGA, AGG, CGG, TGC, TCC,the combined tRNA fragment and the pTRNA_backbone were digested using the restriction enzymes EcoRI and PstI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning.	Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA_backbone.
Week 37 09/09/2019-09/15/2019	Cloning of tRNA fragments into ptRNA_backbone	Katharina Kocher; Benedikt Schober	After a re-synthesis of the ptRNA_backbone, the tRNA fragments AGA, AGG, CGG, TGC, TCC,the combined tRNA fragment and the pTRNA_backbone were digested using the restriction enzymes EcoRI and PstI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning.	Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA_backbone.
Week 38 09/16/2019-09/22/2019	Cloning of tRNA fragments into ptRNA_backbone	Katharina Kocher; Benedikt Schober	After a re-synthesis of the ptRNA_backbone, the tRNA fragments AGA, AGG, CGG, TGC, TCC,the combined tRNA fragment and the pTRNA_backbone were digested using the restriction enzymes EcoRI and PstI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning.	Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA_backbone.
Week 39 09/23/2019-09/29/2019	gDNA Isolation	Benedikt Schober	The tRNA fragments AGA, AGG, CGG, TGC, TCC were amplified from the Vibrio natriegens DSM 759 genome chr.1.	The PCR was succesfully checked by agarose gel electrophoresis.
Week 40 09/30/2019-10/06/2019	Cloning of tRNA fragments into ptRNA_backbone	Katharina Kocher; Sabrina Holl	Cloning of the tRNA fragments into the ptRNA_backbone was performed using Gibson Assembly. Overhangs were introduced by PCR and Gibson assembly was performed according to the NEB Gibson Assembly protocol.	Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA_backbone.
	Mutagenisis of sfGFP, improvement of Gfp and sfGfp biobricks for vibrio natriegens	Katharina Kocher, Sabrina Holl, Hannah Berreth, Verena Haug	Tags of rare Codons (AGA, AGG, CGG, TGC, TCC) were introduced into the sfGFP vector using PCR. The sfGFP with tags of rare codons was transformed into DH5α.	Following transformation into DH5α revealed no successful cloning. No colonies were obtained after transformation.
Week 41 10/07/2019-10/13/2019	Improvement of Gfp and sfGfp biobricks for vibrio natriegens	Hannah Berreth, Verena Haug
Week 42 10/14/2019-10/20/2019	Improvement of Gfp and sfGfp biobricks for vibrio natriegens	Hannah Berreth, Verena Haug
	Expression of improved GFP and sfGFP in Vibrio natriegens	Marvin Bubeck, Jan Müller
	Cloning of tRNA fragments into ptRNA_backbone	Katharina Kocher; Sabrina Holl	Cloning of the tRNA fragments into the ptRNA_backbone was performed according to the NEBuilder HiFi DNA Assembly protocol.	Following transformation into DH5α revealed no successful cloning. No colonies were obtained.
	Transformation of pRARE plasmid in Vibrio natriegens and growth curves	Katharina Kocher; Sabrina Holl	The pRARE plasmid was prepared from E.coli Rosetta and transformed into Vibrio natriegens. Growth curves of the wild type Vibrio natriegens and Vibrio natriegens cells with pRARE were performed.	Growth curves reveald no differences in growth velocity between wild type Vibrio natriegens and Vibrio natriegens cells with pRARE.
	Transformation of sfGFP containing tags of rare codons into Vibrio natriegens.	Katharina Kocher; Sabrina Holl	Tags of rare Codons (AGA, AGG, CGG, TGC, TCC) were introduced into the sfGFP vector using PCR. The sfGFP with tags of rare codons was transformed into Vibrio natriegens.	Following transformation into Vibrio natriegens revealed no successful cloning. No colonies were obtained after transformation.
	competent cells Vibrio natriegens DSM759	Jannis Stadager, Dimitri Graf	competent Vibrio natriegens DSM 759 were made according to iGEM Marburg 2018	Competent vibrio natriegens DSM 759 cells were checked on performance via transformation of the pRARE plasmid performed by Katharina Kocher and Sabrina Holl

Vibrio - Biobrik

Week	Experiment	Participents	Description	Results
Week	Experiment	Participents	Description	Results
Week 40 09/30/2019-10/06/2019	Biobrick charakterisation	Sini Münßinger, Katharina Hofer, Jan Seeger	Plamsid DNA was transformed in E. coli MG1655 via electroporation.
Week 41 10/07/2019-10/13/2019	Biobrick charakterisation	Sini Münßinger, Katharina Hofer, Jan Seeger	For the characterisation, E. coli was cultivated and the RNA purificated by Zymo Quick RNA. During the cultivation, mRFP fluorescence was measured. qPCR was performed to quantify the mRNA amount of mRFP.
Week 42 10/14/2019-10/20/2019	Biobrick charakterisation	Sini Münßinger, Katharina Hofer, Jan Seeger

Vibrio - qPCR

Week	Experiment	Participents	Description	Results
Week	Experiment	Participents	Description	Results
Week 26 06/24/2019-06/30/2019	Vibrio natriegens glycerol stocks	Katharina Hofer	Based on a lyophilized Vibrio natriegens pellet, glycerol stocks were produced.
Week 27 07/01/2019-07/07/2019
Week 28 07/08/2019-07/14/2019
Week 29 07/15/2019-07/21/2019
Week 30 07/22/2019-07/28/2019
Week 31 07/29/2019-08/04/2019
Week 32 08/05/2019-08/11/2019	RNA purification of Vibrio natriegens wildtyp	Sini Münßinger, Katharina Hofer, Jan Seeger	Comparison of two different RNA purification kits: Zymo Quick RNA (Zymo Research) and Nucleospin miRNA (Macherey &Nagel;kit is able to purificate small RNA (>200nt).
Week 33 08/12/2019-08/18/2019	First execution of whole protocol	Sini Münßinger, Katharina Hofer, Jan Seeger	The whole protocol was carried out for the first time. RNA purificated by both kits was used. qPCR was performed with KAPA mastermix. qPCR with the cDNA based on the M&N RNA was performed again, PCR products were sequenzed by Eurofins.	Melting curve showed several amplification products based on the RNA of Zymo kit. RNA purificated by M&N kit will used for further experiments because the meltling curve showd only one amplification product. Each tRNA species showed different ct-values. PCR products were loaded on a gel, but the amplificates were shorter than the expected length. Results of sequencing showed no amplification of the complete sequence (adapter and tRNA).
Week 34 08/19/2019-08/25/2019	qPCR with GreenMasterMix	Sini Münßinger, Katharina Hofer, Jan Seeger	Ligation and reverse transcrition were performed. GreenMasterMix (Genaxxon) was used for the qPCR. qPCR products were sequenzed again.	Each tRNA species showed different ct-values. PCR products were loaded on a gel, but the amplificates were shorter than the expected length. Results of sequencing showed no amplification of the complete sequence (adapter and tRNA).
Week 35 08/26/2019-09/01/2019	qPCR with GreenMasterMix	Sini Münßinger, Katharina Hofer, Jan Seeger	Ligation and reverse transcrition were performed. GreenMasterMix (Genaxxon) was used for the qPCR.	Each tRNA species showed different ct-values. PCR products were loaded on a gel, but the amplificates were shorter than the expected length.
Week 36 09/02/2019-09/08/2019	Comparison of tRNA concentration after different times of cultivation	Sini Münßinger, Katharina Hofer, Jan Seeger	Vibrio was cultivated as usual, samples were taken after 1,5h (mid exponential phase) and 3h (beginning stationary phase). RNA purification and protocol as usual. In addition purified E.coli tRNA (Merck) was treated as usual to verify the protocol.	No differences between the samples of 1,5h and 3h regarding the ct-values. PCR products were loaded on a gel, but the amplificates were shorter than the expected length.
Week 37 09/09/2019-09/15/2019	Comparison of tRNA concentration after different times of cultivation	Sini Münßinger, Katharina Hofer, Jan Seeger	Vibrio was cultivated as usual, samples were taken after 1,5h (mid exponential phase) and 3h (begin stationary phase). RNA purification and protocol as usual. In addition purified E.coli tRNA (Merck) was treated as usual to verify the protocol.	No differences between the samples of 1,5h and 3h regarding the ct-values. PCR products were loaded on a gel, but the amplificates were shorter than the expected length.
Week 38 09/16/2019-09/22/2019		Sini Münßinger, Katharina Hofer, Jan Seeger
Week 39 09/23/2019-09/29/2019	Dilution experiment, preparation of E. coli experiment	Sini Münßinger, Katharina Hofer, Jan Seeger	Verification of the possibilty to determine different tRNA concentrations. Purified V. natriegens tRNA was diluted 1:1, 1:10, 1:100. Sample preparation and qPCR protocol as usual. E. coli Bl21 DE3 and E. coli Bl21 DE3 pRARE were cultivated in 2YT-medium. Cells were harvested after 2,5h, 3,5h and 5,5h and stored at -70°C.	Differences between tRNA concentrations are detectable by ct-value.
Week 40 09/30/2019-10/06/2019	Dilution experiment, comparison of Ecoli BL21 DE3 and pRARE	Sini Münßinger, Katharina Hofer, Jan Seeger	Diltion experiment was performend with all tRNA species in triplicates. Samples were pipetted by Dispendix. Comparison of tRNA concentration of Ecoli BL21 DE3 and BL21 DE3 pRARE.	Differences between tRNA concentrations are detectable by ct-value. No differences between E. coli BL21 DE3 and BL21 DE3 pRARE.
Week 40 09/30/2019-10/06/2019	Implementation of Hotscriptase,	Sini Münßinger, Katharina Hofer, Jan Seeger	Because of the implementation of Hotscriptase (Genaxxon) a new protocol was developed. Treatment of all tRNA species of V. natriegens and Ecoli BL21 DE3 and BL21 DE3 pRARE with new Hotscriptase protocol.	Each tRNA species showed different ct-values. PCR products were loaded on a gel, but the amplificates were shorter than the expected length. qPCR showed different ct-values for the tRNA species of E. coli BL21 DE3 and BL21 DE3 pRARE. The results obtained were not as expected.
Week 41 10/07/2019-10/13/2019	Cultivation and tRNA quantification of E. coli Rosetta	Sini Münßinger, Katharina Hofer, Jan Seeger	tRNA concentation of E. coli BL21 DE3 wildtype, pRARE and Rosetta was quantified and compared via qPCR.	qPCR showed different ct-values for the tRNA species of E. coli BL21 DE3, BL21 DE3 pRARE and Rosetta. The results obtained were not as expected.
Week 42 10/14/2019-10/20/2019

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_t values is the same as in the option described before.

References

1. 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

 

2. 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

 

3. 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

 

4. 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

 

5. 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

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

1. 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.
2. T.L. Babayan, M.G. Bezrukov, “Autolysis in yeasts”, Acta Biotechnol, 5 (2) (1985), pp. 129-136.
3. 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.
4. Takeda, “Classification of Chlorella strains by cell wall sugar composition” Phytochemistry, vol. 27, 12, (1988), pp. 3823-3826.
5. [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.