Fig. X. Restriction of cPCR product representing tyrP knockout generation. tyrP gene have been amplified from genomic DNA and restricted by BcuI. 1212 bp fragments represent wild type genotype, 1014 bp and 184 bp - knockouts. 1 - negative control - no bacteria/DNA added, 2 - tyrP knockout (1), 3 - tyrP knockout (2), 4 - tyrP knockout (3), 5 - tyrP knockout (4), 6 - tyrP knockout (5), 7 - tyrP knockout (6), 8 - tyrP knockout (7), 9 - tyrP knockout (8), 10 - tyrP knockout (9), 11 - tyrP knockout (10).

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).

Fig. X. Comparison of VapXD kill-switch with different promoters before toxin (without bile salts in 24 °C).

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:

Fig. X. Chromatogram at 309 nm. Naringenin control detection by HPLC-MS.

Fig. X. Sample chromatogram at 254 nm. p-coumaric acid control detection by HPLC-MS with retention time 6.165 min.

Fig. X. Sample chromatogram at 309 nm. p-coumaric acid control detection by HPLC-MS with retention time 6.166 min.

This information enabled us to search for compounds in more complex mixtures, in particular LB medium from overnight cultures. Furthermore, we were able to distinguish our products based on retention time, m/z and UV-Vis absorption spectrum.

Using the HPLC-MS method we analyzed the media samples of cultures containing pTRKH2 vectors with TAL gene and J23101 Anderson or surface layer protein A (slpA) promoters. The data of the experiments showed that plasmid with J23101 Anderson promoter and TAL encoding sequence determines an efficient synthesis of p-coumaric acid in our transformants, nevertheless analogous process has not been identified in the samples containing slpA promoter. We hypothesized that the reason for this data non-reproducibility may be a possible mutation in pTRKH2 vector containing slpA promoter, the hypothesis later was approved by Sanger sequencing.

Similar intermediate compound detection strategy was applied for constructs containing 4CL and CHS encoding sequences. We supplied cultures with p-coumaric acid as a substrate for their specific reactions and used HPLC-MS method to detect the consumption of p-coumaric acid. The experiments were conducted with cultures containing pTRKH2 vectors with 4CL gene and J23101 Anderson or slpA promoters, as well as linked (linkers: GSG, GGGGS, (GGGGS)2, (GGGGS)3, EAAAK, (EAAAK)2, (EAAAK)3) 4CL and CHS genes under the same promoters. However, none of the aforementioned constructs have been found to demonstrate distinct enzymatic activity by consummation of the given substrate.

In the hopes of finding further intermediates we searched for a few additional m/z as a result of accumulation. p-coumaroyl-CoA and naringenin chalcone were chosen as the ones who could give us more information. However, fusion protein samples did not show any signs of naringenin chalcone. Moreover, we found the same m/z of p-coumaroyl-CoA in both control and sample from the desired construct medium when supplied with additional p-coumaric acid. This suggested to us that we could not precisely determine the quantity of synthesized p-coumaroyl-CoA because we do not know detailed information about internal processes. We hypothesize that control E. coli DH5α has 4CL homology enzymes for forming carbon-sulfur bonds as acid-thiol ligases and thus synthesis of p-coumaroyl-CoA by both recombinant and native enzymes overshadow one another. We even cannot be sure about synthesis of a particular compound because it needs further analysis by NMR as chromatograms and UV-Vis spectrum lack structural information.

Detailed reports from HPLC-MS are referred to in table X.

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.

Fig. X. Initial testing with ePCR.

ePCR v1

ePCR v1

We observed produced micelles using fluorescent microscopy (400x Magnification) with purified GFP shown in figures X and X.

Fig. X. Fluorescence microscopy with GFP in micelles.

Fig. X. Fluorescence microscopy with GFP in micelles.

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.