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.

Growth curves Vibrio natriegens

Results

Vibrio natriegens was cultivated and samples were taken as described (Protocol_Growth_Curve_Vibrio_natriegens.pdf). The results are shown in figure 1. V. natriegens grew with a maximum growth rate of µ_max= 2,45 h^-1  and reached a doubling time of t_d = 16,99 minutes.

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).

Outlook

Regarding all experiments, several different cloning methods did not result in successful insertion of tRNA fragments in ptRNA_backbone or pSB1C3. Therefore, we commissioned Synbio-Technologies to perform the cloning. After three weeks the company was also unable to perform the cloning successfully. Since the company has newly synthesized the tRNA inserts and the ptRNA_backbone there should be no error in the DNA sequences and cloning. One explanation for unsuccessful cloning could be the toxicity of the tRNA constructs. Only cells that have taken up the ptRNA_backbone without insert survive and result in colonies due to spurious transcription. Impaired cell function can be due to de-repression of spurious transcription in bacteria. ¹ Constitutive transcription initiation can lead to titration of RNA polymerase and global downregulation in host gene expression. ²

For future experiments one conceivable approach would be the genomic integration of tRNA genes in order to increase the tRNA concentration without a plasmid system.

References

1. Wade JT, Grainger DC. Spurious transcription and its impact on cell function. Transcription. 2018. doi:10.1080/21541264.2017.1381794
2. Lamberte LE, Baniulyte G, Singh SS, et al. Horizontally acquired AT-rich genes in Escherichia coli cause toxicity by sequestering RNA polymerase. Nat Microbiol. 2017. doi:10.1038/nmicrobiol.2016.249

Transformation of pRARE into Vibrio natriegens

As an alternative to cloning our self-designed ptRNA_backbone containing tRNAs of rare codons into Vibrio natriegens we used the pRARE plasmid. It carries genes for several rare tRNAs in Escherichia coli, which allows their concentration to be increased. The pRARE plasmid was prepared from E. coli Rosetta and transformed into Vibrio natriegens (Protocol_Transformation.pdf). Growth curves of the wild type Vibrio natriegens and Vibrio natriegens containing pRARE were performed (Protocol_Growth_Curve_Vibrio_natriegens.pdf). The growth curves represented in Figure 1 show no differences in the growth velocity between both Vibrio natriegens strain variants. Thus, the enhanced expression of tRNAs has no obvious negative effect on the metabolic activity of Vibrio natriegens cells.

In order to investigate the possible positive effects of increased tRNA expression on protein expression, special sfGFP variants were supposed to be developed as explained in section Mutagenesis of sfGFP.  

Mutagenesis of sfGFP

The vector pYTK047 containing sfGFP-Sequence was kindly provided by iGEM Team Marburg. To evaluate the expression of proteins containing rare codons (AGA, AGG, CGG, TGC, TCC) the tags of rare codons were introduced into the pYTK047. During the expression, the tags are fused to the N-terminus of the protein sfGFP. Thereby they determine the initial translation velocity of the expression of the fusion protein.

To insert the tags of rare codons into the pYTK047 genome, PCR with specialized primers was performed (for more detailed information see Q5_Site-directed_mutagenesis.pdf). To remove the unamplified template vector the PCR reaction was digested with Dpn1 and separated by agarose gel electrophoresis (Protocol_Agarose_Gel.pdf). Looking at Figure 1 only the constructs containing the tags AGA (1), AGG (2) and TGC (4) could be amplified by PCR. 

The bands of the desired size were cut out of the agarose gel and the DNA was extracted (Protocol_Gel_Extraction.pdf). A KLD ligation was performed according to the NEB KLD Enzyme Mix Reaction Protocol to circularize the extracted linear DNA. After ligation, the pYTK047 with the tags of rare codons was transformed in E. coli  DH5α following the transformation protocol for chemical transformation (Protocol_Transformation.pdf). No colonies were obtained after transformation. The transformation was repeated, but the plasmid was not transformed successfully. The plasmid was supposed to be isolated from an overnight culture (Protocol_Plasmid_Preparation.pdf) and successful introduction of rare codons into plasmid pYTK047 should have been validated through sequencing. Transformation into Vibrio natriegens (Protocol_Transformation.pdf) and following growth curves or expression analysis should reveal if protein expression is impaired when rare codons are present.

In the last step, we wanted to investigate if the expression of proteins with rare codons can be rescued or even increased when a higher concentration of tRNAs for those rare codons are present. Therefore, pRARE from E. coli Rosetta was supposed to be transformed into V. natriegens together with pYTK047 containing sfGFP with rare codons. The plasmid pRARE codes for the tRNA of proL, leuW, metT, argW, thrT, glyT tyrU, thrU, argU and ileX and therefore increases their concentrations.

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.

1. Media were produced (Protocol_media_first_experiments.pdf).
2. Media were inoculated with escherichia coli or vibrio natriegens.
3. 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.

1. Media were produced (Protocol_media_first_experiments.pdf).
2. Media were inoculated with vibrio natriegens.
3. 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).

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).

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.

Results: Cell wall disruption of C. vulgaris and C. sorokiniana

For cell wall disruption of C. vulgaris and C. sorokiniana physical and chemical approaches were tested. One physical approach was bead milling with different amounts and sizes of glass beads. Also, one experiment was performed with beads with sharp edges. As an chemical approach for cell wall disruption, algae were boiled in acid or base at 100 °C for one hour (https://2019.igem.org/wiki/images/e/ec/T--Stuttgart--Protocol_Cell_extraction_with_autolysis_combined_with_bead-milling.pdf).

To quantify the amount of protein, received from the respective cell wall disruption method, Bradford-Assay (https://2019.igem.org/File:T--Stuttgart--Experiments_BradfordAssay.pdf) was performed. The amount of protein was related with the amount of used biomass. First we decided to test physical cell wall disruption (Fig. 1).

The highest amount of protein (15.6 %) was achieved with 80 % of glass beads with a size between 200-300 µm. The smaller glass beads reached up to 14.04 % of protein and were less efficient. A drastic difference can be seen between 50 % glass beads and 80 % glass beads. With the smaller beads the amount of glass beads used led to a difference of 11.93 %, similar to the bigger beads with a difference of 11.77 %. Therefore, 80% of glass beads was used for future cell wall disruption to create our PhyCoVi medium. Also, the bigger beads led to a higher yield of protein and were therefore chosen to be the future method for cell wall disruption.

An additional approach of physical cell wall disruption was to use special beads with sharp edges at different speeds (Fig. 2). The beads had a size of 2 mm.

The highest yield of protein was gained with C. vulgaris at speed 8. Speed 4 always led to a lower yield of protein. C. sorokiniana reached a lower yield of protein with 1.19 %. The sharp-edged beads reached a lower yield of protein compared to the glass beads with a size of 200-300 µm. Hence, this method was not considered for further use.

To further optimate the cell wall disruption methods and increase the amount of usable sample, cell wall disruption with acid and base was tested. As bases we used 1 M KOH, 5 M KOH and 1 M NaOH, as acid 1 M HCl was used (Fig. 3).

The highest yield of protein was reached with 5 M KOH with 9.03%. Lower amounts of protein were gained with 1M HCl and 1M KOH. NaOH led to the least yield of protein with 3.59 %.

After boiling the solutions had to be neutralized. Therefore, we used 1 M NaOH and 1 M HCl. Neutralizing HCl with NaOH leads to the formation of NaCl, which is a compound of the common cultivation medium for V. natriegens. Our PhyCoVi medium was primary created to be a medium for v. natriegens. Therefore, we chose cell disruption with 1 M HCl to be the future method.

All samples were also analyzed by the Anthrone Method (https://2019.igem.org/wiki/images/f/f3/T--Stuttgart--Experiments_AnthroneAssay.pdf), to quantify the amount of free sugars and starch. The anthrone reagent coagulates with sugars and starch. The more carbohydrates are available, the greener the samples appears and the higher the absorbance at 620 nm. Concerning different sizes of the glass beads, the small beads with 100 to 200 µm showed the best results. But also the high speed disruption with sharp edged glass beads around 2.0 mm showed good results (Fig. 4). Since this option is only available at the Max-Planck-Institute in Tübingen, we chose the options we had available in Stuttgart.

For the two volume ratios of glass beads to sample, the 80 % of 200 µm glass beads showed the best result. Generally more and bigger glass beads show better cell disruption.

At last the acid and base disruption was examined. As seen in Fig. 6, all bases show low values, with 5 M KOH being unusable. With HCl reaching a similar level like glass beads, we chose this acid as our method for cell disruption. In combination with glass bead disruption, we hoped to have an efficient method for cell disruption of micro algaes.

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.

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

Results preliminary Chlorella Vulgaris extraction methods (FACS analysis)

The obtained samples were measured at a flow-cytometer for green fluorescence (intact Chlorophyll) as well as cell size (SSC and FSC). A distinct amount of liquid (50 µL) was measured. Based on the cell size (intact cells show a clear population) and green fluorescence, viable cells were able to be gated into a distinct population. With obtaining a distinct number of viable cells within a constant volume we were able to determine the number of viable cells per µL. The number of cells/µL of the control sample was set to 100% with the extraction methods (high-pressure-homogenizer passages 1 - 4, mortar, bead-mill, autoclave and sonification) set in relation of the control. We hereby obtained the percentage of viable cells for each cell disruption method (Figure 1).

Conclusion:

In terms of feasibility and up-scaling problems the method we further wanted to analyze was the bead-mill. With a 50% cell viability it proved to be a rather solid method for cell disruption. We concluded that further research for the optimal bead size should be conducted as well as a combination with an autolytic process. The idea was that acid induced autolysis prior to the bead-mill treatment should weaken the strong microalgae cell wall and therefore decrease cell viability.

Growth Curves of various bacteria on PhyCoVi Medium

The PhyCoVi medium was tested on various microorganism including Vibrio natriegens, Escherichia coli, Bacillus subtillis and Corynebacterium glutamicum. Therefore, absorption was measured at 600 nm with a photometer for 315 to 316 minutes. Different medium variants were tested to find the best medium composition. For control also normal LB medium, Brain-Heart-Infusion medium and medium containing only soy peptone was tested. Medium composition is shown in Table 1.

To estimate the optimal medium composition for V. natriegens different NaCl concentrations were tested as well as the addition of soy peptone (Figure 1).

The PCV medium containing additional soy peptone revealed the highest growth of V. natriegens. It even outcompeted the commonly used BHIN medium. V. natriegens cultivated in PCV medium without additional soy peptone showed remarkably less growth. LB and Soy peptone alone showed the least bacterial growth.

Next, bacterial growth of Escherichia coli MG1655 on PhyCoVi medium was tested (Figure 2).

Highest bacterial growth was reached with PCV medium containing additional soy peptone and 10 g/L NaCL followed by the 20 g/L NaCL variant. The differences in E. coli growth on various media is not as clear as it was with V. natriegens (Figure 1). Also, the lag phase takes more time. Nevertheless, PhyCoVi medium with additional soy peptone is applicable on cultivation of E. coli.

Further, Growth of Bacillus subtilis (Figure 3) and Corynebacterium glutamicum (Figure 4) were tested. Only medium containing 10 g/L NaCL was used.

Bacillus subitilis grew best on BHIN followed by LB medium. PCV medium with soy peptone was still better than PCV without it. Nevertheless, there was not a remarkably increase in absorbance and thus only little growth of bacteria after 6 h.

Corynebacterium glutamicum grew better on BHIN and LB than on PCV with or without soy peptone. PCV with additional soy peptone showed higher growth than medium without soy peptone. Comparing Bacillus subtilis with Corynebacterium glutamicum, C. glutamicum shows better growth on PCV containing soy peptone. Although both bacteria prefer BHIN and LB medium.

Sugar HPLC Results

The measurement was performed with six technical replicates of the algae extract batch PCV#2 from Chlorella vulgaris. The molar concentration of glucose, fructose, sucrose and raffinose were determined. The results of the carbohydrate HPLC measurement are shown in Table 1.  

We were able to measure glucose and fructose in our samples. Raffinose was not detected at all. Sucrose was measured only in one sample, so we decided to reject its concentration.

By calculating the averages of the 6 replicates we were able to determine the amount of certain carbohydrate in gram per 100-gram Chlorella vulgaris extract. In 100 g C. vulgaris extract we obtain 1.264 g glucose and 0.391 g fructose.

Amino acid HPLC Results

For this experiment we measured three technical replicates of two batches of our self-made algae extract from Chlorella vulgaris with two concentrations 30 g/L and 60 g/L. The two measurements were performed to have a detectable amount of amino acids. Histidine in batch PCV#3 was only measured in the 60 g/L sample, he corresponding values were divided by 2 and added to the 30 g/L values. We calculated averages and standard deviations of the molar concentrations from the HPLC data for all 20 proteinogenic amino acids. To be able to compare the two batches PCV#2 and PCV#3, we plotted the average concentrations in a bar diagram. We were not able to distinguish between the peaks of alanine and arginine. For that reason, they were analyzed as one peak. 

Figure 1: Bar diagram of average molar concentration of 20 proteinogenic amino acids from two batches of self-made algae extract from Chlorella vulgaris diluted in water (30g/l). In blue the prior extract with the ID: PCV#2. In green the final batch with ID: PCV#3.  

https://2019.igem.org/wiki/images/4/4b/T--Stuttgart--FigureHPLC2.png

It is noteworthy, that the batch PCV#3 shows a brighter spectrum of amino acids, than the prior batch PCV#2. When harvesting the algae for the batch PCV#3, we tried to optimize the protocol for extraction. The prior extraction protocol had some issues with product purity. The powder contained some disintegrated algae contaminants. However, we could achieve an improvement when it comes to amino acid content. 

To create a content list for our algae C.vulgaris extract, we calculated the mass of amino acids per 100 gram extract for both batches PCV#2 and PCV#3. The corresponding list is shown in the following table.

https://2019.igem.org/wiki/images/0/09/T--Stuttgart--FigureHPLC3.png

The total amount of proteinogenic amino acids in PCV#2 is 0.207 g per 100 g extract. In PCV#3 we have a total amount of 0.339 g per 100 g extract. With the optimized protocol we were able to achieve 0.6 times more proteinogenic amino acids per gram extract than before.