Team:Aalto-Helsinki/Demonstrate
RESULTS
Our laboratory work started with the aim of demonstrating the usefulness of Tat pathway of Vibrio natriegens in protein production and secretion.
For this purpose we needed to show that we have identified a working signal sequence, attached it to a protein of choice, and demonstrated that the protein translocates to the periplasm with the signal sequence.
After establishing that Tat pathway of Vibrio natriegens can be utilized in protein translocation, we determined a series of modifications that we’d use to show that the protein yield can be increased by the overexpression of Tat complexes in the cytoplasmic membrane, in a similar fashion to the TatEXPRESS strain of Browning et al. (2017).
According to Browning et al. and our research, there are two useful strategies for this: the Tat subunits can be expressed from a plasmid vector while the constitutive Tat expression in the chromosome is active, or the expression of the subunits can be induced from the chromosome, by placing inducible promoters upstream of the native ones.
After demonstrating this, we planned to further modify the genome by several deletions to further improve the possible protein yield, and establish an expression strain, VibXPresso. Unfortunately, in iGEM like in life generally, time is of the essence. While VibXPresso remains under construction, our results demonstrate the viability of our results demonstrate the viability of our fundamental theory, by showing that the putative signal peptide functions correctly, and translocates YGFP reporter to the periplasm in Vibrio natriegens.
Cloning was performed using traditional restriction endonucleases and blunt-end ligation to the vector. Before digestion, appropriate restriction enzyme cut sites were added to the fragments by PCR (see Protocols).
Photo 1: Successful PCR of the plasmids pC201 and pC203 to remove the PluR insert. dPRha denotes a version of the plasmid where the rhamnose promoter has also been removed, to possibly test and compare V. natriegens’ native rhamnose promoter to the E. coli one.
Photo 2: Amplified DNA fragments of ssTorA-YGFP, ssAminotransferase-YGFP, ssTorA-hGH and ssAminotransferase-hGH with modified restriction enzyme cut sites, to clone into pC201 and pC203 vectors.
Most of the digestion reactions were performed with Thermo Fisher Scientific’s Fast Digest enzymes EcoRI and XbaI, cut sites of which were added to the plasmid backbones during the PCR reaction. Ligations were performed subsequently with Thermo Fisher Scientific’s T4 DNA Ligase. The cells were then transformed and insert including transformants determined with colony PCR.
Photo: Digestion reaction result for ssTorA-hGH and pC203-ssTorA-YGFP plasmid. Digested ssTorA-hGH on lane 2 from the left, on lane 3 undigested control. Digested pC203-ssTorA-YGFP on lane 4, lower band being the cut ssTorA-YGFP fragment.
Photo 3: cPCR screening of ssTorA-YGFP inserts in pC201 and pC203 backbones. PC201 exhibits variation that may have been a reason for pretty poor results in expression tests. Most of the pC203 constructs have the correct insert. Also up can be seen just for fun made pC201 and pC203 digestions. The amount of DNA was not enough to be clearly visible in gel.
Photo 4: cPCR screening of ssTorA-hGH. Most of the colonies have the correct insert.
Photo 5: cPCR screening of pC203-tfoX, with most of the colonies having the correct insert.
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Total constructs assembled:
- pC203-ssTorA-YGFP
- pC203-ssTorA-hGH
- pC203-ssAminotransferase-YGFP
- pC203-hGH (to serve as control in fractionation experiment)
- pC203-tfoX (for chromosomal editing)
- pC201-ssTorA-YGFP
- pC201-ssAminotransferase-YGFP
Expression of ss-YGFP from pC203 with a range of L-rhamnose concentrations indicated that expression was strongest with highest L-rhamnose concentrations tested. This was observed both in Vibrio natriegens and E. coli DH5α. Absolute fluorescence values were higher in DH5α compared to V. natriegens. This was most likely due to the fact, that the expression system was not optimized for V. natriegens at this stage. With both bacteria, highest fluorescence values were measured after 3,5 h. Data for induction of pC201-ss-YGFP is not shown, because fluorescence readings were significantly lower indicating the construct was not functioning properly.
Graph showing fluorescence of YGFP measured at 500-530 nm in V. natriegens over 10 h. A range of L-rhamnose concentrations were tested to understand the sensitivity of our expression vector. Fluorescence data was normalized with OD600 values measured at the same time points.
Graph showing fluorescence of YGFP measured at 500-530 nm in DH5a over 10 h. A range of L-rhamnose concentrations were tested. Fluorescence data was normalized with OD600 values measured at the same time points.
After o/n induced expression with 25 mM L-rhamnose, Vibrio natriegens cells were imaged using Leica DM5000B fluorescence widefield microscope, at the Light Microscopy Unit of the Institute of Biotechnology at the University of Helsinki. The photos were taken with Hamamatsu Orca-Flash 4.0 V2 sCMOS camera.
Fluorescence image displays polar YGFP localisation in the periplasm of V. natriegens cells.
This was also confirmed with fractionation of the cells, where YGFP was clearly visible in the periplasmic fraction under UV lights (see photo below).
GFP as a reporter has been extensively studied, and the localisation to cell poles with a TorA signal sequence is a known phenomenon, that may be caused by the growth conditions and change in the environment, which affects the topography of the periplasmic space (Santini et al., 2001; Sochacki et al., 2011). In certain expression conditions, the TorA signal peptide has also been shown to direct proteins into inclusion bodies in E. coli (Jong et al., 2017), but in our case this seems unlikely as the expression levels remained low. However, the observation demonstrates that the signal peptide is functional and protein translocation or directing happens in V. natriegens.
Photo: Periplasmic fractions of V. natriegens showing the YGFP localisation in the periplasmic fraction. Tubes on the left contain whole cells in growth medium as a reference.
Somewhat similar polar localisation was also observed in some E. coli DH5α cells after induction with 5 mM L-rhamnose. Since the required double arginine RR motif is also found in the TorA signal peptide of Vibrio natriegens, the translocation mechanism may well be active in E. coli as well. Another explanation might be the inclusion bodies, but a well established conclusion on this would require more experiments and testing with DH5α cells.
Photo: E. coli DH5α cells expressing the ssTorA-YGFP gene. A polar localisation of the YGFP is visible in some of the cells.
We built a similar plasmid construct with ssTorA-hGH insert, to test its localisation in V. natriegens cells. After transforming the constructs to both V. natriegens and E. coli DH5α, we screened the successful transformants with PCR and continued to expression tests. The C-terminus of hGH included a hexa His tag, which enables easy purification to detect (and later measure) the amounts of hGH in the cytoplasm, periplasm, and insoluble fraction.
The construct was induced similarly to the ssTorA-YGFP, with 25 mM L-rhamnose. After a 3,5 hour expression period, the cells were pelleted and fractionation to cytoplasmic, periplasmic, and insoluble fraction performed according to the modified PureFrac protocol (see Protocols). The resulting fractions were run in SDS-PAGE and blotted to nitrocellulose membrane for His detection. The detection gave no results, and the reason was clarified soon after. After sequencing our ssTorA-hGH constructs, turned out that the hGH had mutated in the cells, with numerous nucleotide changes and deletions. Also the gene was truncated so that the beginning had disappeared (together with ssTorA) in all of our sequenced samples. Since the construct had been purified from Vibrio natriegens cells, it might be that the organism has attacked our gene due to its toxicity. Continuing will require tests with different codon optimizations to see if we’ll manage to get the product expressed.
Photo: Blot of the unsuccessful hGH detection.
Standard curves and equations used for calculating dxs and bla gene copies are presented below. Dxs gene copies were calculated with the equation y = -1,457ln(x)+39,763 and bla gene copies with y= -1,444ln(x)+39,809.
The amount of dxs gene copies and bla gene copies in V. natriegens harboring pQE30 vector was 1,0 x 10^6 and 2,9 x 10^6 copies respectively. Copy numbers in V. natriegens with pUC18 vector were 1,7 x 10^6 for dxs and 7,7 x 10^6 copies/ul. V. natriegens harboring pC203 vector had 1,1 x 10^6 copies/ul of dxs gene and 6,6 x 10^6 copies/ul of bla gene.
Dxs gene copies quantified from total DNA extracts from V. natriegens. Bars represent mean values of three biological replicates and error bars represent standard deviations. NTC = no template control.
Bla gene copies quantified from total DNA extracts V. natriegens. Bars represent mean values of three biological replicates and error bars represent standard deviations. NTC = no template control.
Copy numbers of pQE30, pUC18, and pC203 plasmids (ColE1, pMB1 and p15A origins of replication respectively) in V. natriegens were 3,0; 45,2 and 6,2 copies/gDNA. In a recent study, copy numbers of plasmids with same origins of replication were quantified in V. natriegens in a similar fashion (Tschirhart et al., 2019). However, copy numbers were higher compared to our results for ColE1 and pMB1 origins of replications (313 copies/gDNA and 106 copies/gDNA respectively). Copy number of p15A was similar to our result (5 copies/gDNA). A possible explanation for the differences could be the OD-value, to which cells were cultured before harvesting total DNA. In a protocol by Lee et al. (2006) cells were in exponential growth phase (OD600 = 0,5), whereas Tschirhart et al. (2019) harvested cells at stationary phase. In our experiment, pQE30 and pUC18 (ColE1 and pMB1 oris) where harvested at exponential growth phase and pC203 (p15A ori) at stationary phase.
Copy numbers of pUC18, pQE30 and pC203 plasmids (pMB, p15A and ColE1 origins of replication respectively). Bars represent mean values of three biological replicates and error bars represent standard deviations.
We performed PCR for pC203 with our designed primers PC20x_dPluR/dProm fwd & PC203_dPluR rev (anneal T= 62.3C) to remove PluR, and confirmed with electrophoresis gel that we had successfully performed PCR, pC203 without PluR = 4880bp.
We successfully performed PCR and confirmed results with an electrophoresis gel showing a band at 600bp for tfoX amplification.
We screened for successful inserts using primers Tfox fwd & PC203_insert_conf rev (product = 690bp, anneal T =59,9C)
tfoX-pC203 construct
For the planned dns deletion, we performed PCR to amplify the upstream region for homologous recombination vector using our designed primers Dns 2.6k upstream fwd & Dns OE cfwd-rev (anneal T= 62.7C), and the downstream region for homologous recombination vector using primers Dns 2.5k downstream rev & Dns OE crev-fwd (anneal T= 62.0C).
For the planned tat deletion, we also performed PCR to amplify the upstream region for homologous recombination vector using our designed primers TatO 2.54k upstream fwd & TatO OE cfwd-rev (anneal = 61.3), and the downstream region for homologous recombination vector using primers TatO 2.5k downstream rev & TatO OE crev-fwd (anneal T= 61.4C).
Due to time constraints, we decided to continue only with the dns deletion into the next step. In the next step, we combine pieces of upstream and downstream regions by overlap extension PCR (OE-PCR) to form a complete vector (anneal T = 72C).
Next, we performed PCR with our designed primers Dns 2.6k upstream fwd & Dns 2.5k downstream rev to amplify complete vector (anneal T= 62.3C). After, we run an electrophoresis gel of PCR products from and extracted the band at 5000bp.
We then transformed the complete vector into (naturally competent, induced tfoX expression plasmid containing) V. natriegens and plated transformed cells on ampicillin + V2 + LB containing plate.
Next, we picked colonies and attempted to verify deletion by PCR using primers Dns 3.1k upstream fwd & Dns 3k downstream rev (anneal T = 63 C). Finally, we analyzed the product in electrophoresis gel (deletion-positive = 6000bp). However, most likely due to wrongly calculated extension time, we were not able to get a confirmation before running out of time.
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Jong, W. S., Vikstrom, D., Houben, D., van den Berg van Saparoea, H. B., de Gier, J. W., & Luirink, J. (2017). Application of an E. coli signal sequence as a versatile inclusion body tag. Microb Cell Fact, 16(1), 50. doi:10.1186/s12934-017-0662-4
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Santini, C. L., Bernadac, A., Zhang, M., Chanal, A., Ize, B., Blanco, C., & Wu, L. F. (2001). Translocation of jellyfish green fluorescent protein via the Tat system of Escherichia coli and change of its periplasmic localization in response to osmotic up-shock. J Biol Chem, 276(11), 8159-8164. doi:10.1074/jbc.C000833200
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