Team:CityU HK/Results

Results - iGEM2015 wiki

Characterization Improvement Results:

Figure 2. Quantitative RT-PCR analysis of lacZ and lacY transcripts in E. coli DH5α host cells. 
Expression of the lacZ and lacY genes was determined in control DH5α cells (BLUE) and BBa_S04055 recombinant DH5α cells (RED). The cells were cultured overnight in LB medium and harvested for total RNA extraction.

Quantitative real-time PCR

Quantitative real-time PCR analyses showed that both lacY and lacZ transcripts were expressed at high levels in the recombinant E. coli cells as compared to control (Figure 2). The lacY gene expression was 9-fold higher in recombinant cells than the control cells. For the lacZ gene mRNA expression, the recombinant cells were 2-fold higher than the control cells.




Western Blot 

Western blot analysis demonstrated that β-galactosidase protein (indicated by the arrow) was expressed higher in recombinant BBa_S04055 E. coli than the control cells. The result matches that of the qRT-PCR assay, suggesting β-galactosidase was overexpressed in the BBa_S04055 recombinant cells.
Figure 3. Western Blot analysis of the expression of β-galactosidase. 
Protein expression of β-galactosidase in the control cells (Lane 1) and BBa_S04055 transformant (Lane2).
Figure 4. Expression of β-galactosidase as measured by the ONPG assay.
Control (BLUE) E. coli cells and BBa_S04055 recombinant cells (RED) at OD600=0.3 were harvested and lysed to release intracellular β-galactosidase, which was then measured for its activity by the ONPG assay. The conversion was measured by Ab420 at 15 minute interval.

ONPG Assay

The assay showed that the cleavage product increased linearly within 60 minutes in BBa_S04055 recombinant cells and no change in absorbance in  was observed in the control cells, suggesting that LacZ protein was expressed in BBa_S04055 recombinant cells, which matches the result reported by the iGEM 2008 Caltech team.