Team:Vilnius-Lithuania/Results
RESULTS
Prevention
Promoter characterization
To evaluate the quantity of synthesis by our constructs/enzymes, we employed HPLC-MS to find naringenin and intermediate compounds. All enzymes were subjected to analysis first by themselves and further in different combinations. Both control for native cellular metabolism and with additional substrates were taken into account.
mRNA cyclization system evaluation
To evaluate the quantity of synthesis by our constructs/enzymes, we employed HPLC-MS to find naringenin and intermediate compounds. All enzymes were subjected to analysis first by themselves and further in different combinations. Both control for native cellular metabolism and with additional substrates were taken into account.
Metabolic pathway construction
To construct the pTRKH2 vector containing all four genes of the naringenin metabolic pathway we amplified pTRKH2 vector and all four naringenin synthesis genes using primers that contain specific restriction endonuclease recognition sites. This way we should have been able to digest each sequence with appropriate restriction enzymes and create a library of inserts with sticky ends, that can be ligated into the target vector as the ending part of composite insert or as a part of the whole naringenin synthesis cassette. However, we were only able to construct plasmids containing only TAL and TAL+4CL, cassettes that later were found to have been mutated by Sanger sequencing.
To get the construct containing all four genes we chose the strategy of Gibson assembly. By amplifying the pTRKH2 vector and all naringenin synthesis genes with primers containing flanking regions that form homologous pairs with each other in the manner that a complete naringenin synthesis cassette should be constructed in a single tube reaction. Nevertheless, we were not able to obtain the desired construct, only the plasmids.
Kill-switch
To quantitatively evaluate VapXD kill-switch performance, OD600 measurements were performed. First of all, VapD toxin (BBa_K3904000) activity was characterized while regulating its production with cold-induced promoter (BBa_K3904003). Graphs at the top of figure X illustrate bacteria growth without toxin and graphs at the bottom with toxin in different temperatures. While comparing obtained data in 37 and 24°C, temperature change can be seen as a factor inducing greater toxin production and cell death. On the other hand, VapX activity is not fully accurate due to the leakage of the promoter.
Furthermore, VapXD with the bile-induced promoter before antitoxin and with the cold-induced promoter before toxin was characterized. Graphs in the top of X figure demonstrate bacteria growth with and without bile salts supplementation in media at 24 °C, as graphs in the bottom at 37 °C. It can be seen that OD600 in the presence of bile salts and 37 °C bacteria grow more exponentially than without bile salts and in 24 °C. In the ideal case, no antitoxin should be produced in the absence of bile salts and 24 °C, and toxin synthesis should be induced. However, the results indicate that in such conditions, bacteria growth is only slightly repressed.
When results in 37 °C obtained with different OD600 values were averaged (fig. X “Mean comparison with/no bile 37 °C), the difference between measurements with and without bile salts appeared to be mathematically insignificant.
What is more, the activity of different promoters before VapX toxin was compared without bile salts supplementation in media at 37 °C. From promoters’ strength evaluation measurements (fig. X), it was seen that the p-slpA promoter (BBa_K3904712) is the strongest in our inventor. VapXD assessment also showed that under this promoter, toxin production is more significant than under other promoters. The sloping graph rise illustrates this because more toxin is produced, and bacteria growth is inhibited. However, after the results were averaged, no significant difference between different promoters is seen.
While comparing results with different promoters in 24 °C no significant difference can be seen (fig. X).
Genome editing
We seek to create naringenin producing probiotics. For this reason, we decided to insert naringenin metabolic pathway encoding genes into E. coli Nissle 1917 genome. This experimental decision helps to overcome the problem of additional antibiotic usage and reduce the fluctuations gained because of unstable plasmid copy numbers in cells. Firstly, to measure the transcription activity from two genomic regions, we have inserted sfGFP into colicin and nupG genes (fig. X, X) and compared the amount of fluorescence (fig. X).
Furthermore, to enhance naringenin synthesis in E. coli Nissle 1917 we created ackA-pta double knockout. Firstly, we knockouted ackA (fig. X), and pta (fig. X) genes separately. Later on, we used ackA knockout to generate ackA-pta double knockout (fig. X). As we see in (fig. X) sgRNA designed for ackA knockout creation shows 100 % efficiency as all randomly selected colonies had desired changes in ackA gene. For further experiments chosen ackA knockout have been verified by ackA gene sequencing. pta knockout also have been generated with 60 % efficiency (fig. X). In addition, ackA-pta knockout have been created by generating pta knockout from ackA knockout with 80 % efficiency of pta knockout generation.
Naringenin evaluation
To evaluate the quantity of synthesis by our constructs/enzymes, we employed HPLC-MS to find naringenin and intermediate compounds. All enzymes were subjected to analysis first by themselves and further in different combinations. Both control for native cellular metabolism and with additional substrates were taken into account.
First, we created control chromatograms for naringenin and first enzymatic intermediate - p-coumaric acid (product of Tyrosine ammonia lyase (TAL) from naringenin synthesis pathway). By dissolving technical grade compounds in pure water we found retention times:
| Naringenin | p-coumaric acid |
|---|---|
| 6.88 min | 6.17 min |
| Name | Description | File |
|---|---|---|
| Technical grade p-coumaric acid | Technical grade p-coumaric acid was dissolved in water for reference chromatogram and other specifications. | Download |
| TAL1 lysate | First enzyme Tyrosine ammonia lyase (TAL) in pTRKH2 plasmid with supplied p-coumaric acid to LB medium. Culture was lysed to check for compounds inside cells. | Download |
| TAL1 LB medium | First enzyme Tyrosine ammonia lyase (TAL) in pTRKH2 plasmid with supplied p-coumaric acid to LB medium. Only LB medium was subjected to HPLC-MS analysis. | Download |
| DH5alpha with p-coumaric acid | Only DH5alpha without any plasmid cells were grown in LB medium with supplied p-coumaric acid. | Download |
| LB with p-coumaric acid | LB medium with p-coumaric acid. | Download |
| LB without p-coumaric acid | LB medium without p-coumaric acid. | Download |
| GS1 LB medium without p-coumaric acid | Fusion protein (4CL and CHS) construct with GGGGS linker in pTRKH2 plasmid without supplied p-coumaric acid to LB medium. | Download |
| GS1 lysate with p-coumaric acid | Fusion protein (4CL and CHS) construct with GGGGS linker in pTRKH2 plasmid without supplied p-coumaric acid to LB medium. | Download |
| GS1 lysate with p-coumaric acid | Fusion protein (4CL and CHS) construct with GGGGS linker in pTRKH2 plasmid without supplied p-coumaric acid to LB medium. | Download |
| GS1 LB medium with p-coumaric acid | Fusion protein (4CL and CHS) construct with GGGGS linker in pTRKH2 plasmid with supplied p-coumaric acid to LB medium. | Download |
| GSG LB medium with p-coumaric acid | Fusion protein (4CL and CHS) construct with GSG linker in pTRKH2 plasmid with supplied p-coumaric acid to LB medium. Culture was lysed to check for compounds inside cells. | Download |
| Naringenin | Technical grade naringenin dissolved in distilled water. | Download |
Detection
Entamoeba histolytica recombinant protein synthesis
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SELEX
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Emulsion PCR
We found creation of emulsion an easy task, but nonetheless few problems occured. First, version 1 emulsion showed irresistance for thermal cycling and micelles broke even after 15 cycles. For this we tried creating emulsions in colder conditions and by mixing for longer. None of these showed better results. Furthermore, comparison of products between ePCR and oPCR was done. In figure X different cycle count was used and on this basic data we can see that open PCR started generating non-specific fragments after 25 cycles and emulsion PCR lagged at overall production of fragments but in the end did not create same longer fragments as seen in oPCR.
ePCR v1
ePCR v1
We observed produced micelles using fluorescent microscopy (400x Magnification) with purified GFP shown in figures X and X.
Micelles were stable at room temperature while observing them.
ePCR v2
We tested updated composition emulsion and it did not show any signals of breakage even after 50 PCR cycles. The main problem with this is when we want to check nonspecific PCR products in electrophoresis. It is not an easy task to break emulsion with neither 1-butanol, nor isopropanol. However when used in PCR purification kit the emulsion has gone from cloudy to clear from binding buffer and centrifugation at 20.000 rcf.
To recreate both versions of emulsions
Observations
Distribution among PCR tubes by 50 µl leaves quite visible mineral oil smear on pipette tips. It is better to produce more of the overall mixture for higher yield. As per visual examination emulsion breaks even after 10 cycles of PCR which suggests that different emulsifiers should be used.