Team:Vilnius-Lithuania/Results

Prevention

Promoter characterization

mRNA cyclization system evaluation

Metabolic pathway construction

Kill-switch

Genome editing

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

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

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

Fig. X . Description

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.

References

1.

Trundle, K. Teaching Science During the Early Childhood Years. National Geographic Learning (2010).

11.