Difference between revisions of "Team:CityU HK/Results"
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Revision as of 13:22, 17 September 2015
Figure_. 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_). The lacY gene expression was 9-fold higher in recombinant cells than the control cells. For the lacZ gene mRNA expression, the recombinant cells was 2-fold higher than the control cells.
(Any replications on the experiments? N= times of replication) |
Western BlotWestern 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.
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Figure _. 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 _. 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.
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