Team:Hong Kong UCCKE/Assays Results
Assay Results
We carried out two assays to test the presence and effectiveness of the transformed E.coli containing BBa_K3077100. Firstly, we verified the presence of the DNA by colony PCR followed by gel electrophoresis. Secondly, we tested the catalytic activity on Tributyrin Agar of the bacteria.
Assay 1: DNA verification
Aim:
To determine the presence or absence of insert DNA (composite part BBa_K3077100) in 6 plasmid constructs
Method:
Colony PCR followed by gel electrophoresis
Results:
Referring to the DNA ladder, clone 1,2,3,5,6 collectively showed bands at about 1.6 kB, which fits the sequence length of the composite part BBa_K3077100 of 1580 bp, thus verified the presence of the correct insert. On the other hand, clone 4 failed. The E.coli of the correct clones are then amplified and used in the assay below.
Assay 2: Lipid hydrolysis assay
We tested the catalytic activity of the transformed composite part BBa_K3077100 with 91010 Tributyrin agar. E.coli transformed with the composite part plasmid was spread on a Tributyrin agar plate. A negative control without this construct was also streaked out on another plate. The cells were incubated for 1 day.
Results:
Fig.2 Results of clones where that showed less lipid droplets underneath the E.coli
Fig.3 Black and White Results of clones that showed less lipid droplets underneath the E.coli
Of the 5 sets of data, only 2 clones showed the growth of clear zone underneath the E.coli than the negative control, but overall there is no clear positive results. This can be explained by the esterase(Lip8) being cytoplasmic, which makes it inaccessible to its substrate in the external environment unless the substrate passes through the outer membrane, or the esterase leaks out of the cell in a significant amount. Therefore, we plan to carry out the Lipid hydrolysis assay again after cell lysis to test its cytoplasmic catalytic activity. Yet, we can’t do as planned due to insufficient time and it will be continued in our next year project.