Difference between revisions of "Team:CityU HK/Results"

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<h2 class="wsite-content-title" style="text-align:left;">Characterization Improvement Results:</h2>
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<h2 class="wsite-content-title" style="text-align:left;">Improved/ expanded characterization of LacY-LacZ biobrick (BBa_S04055)</h2>
  
 
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Revision as of 07:10, 18 September 2015

Results - iGEM2015 wiki

Improved/ expanded characterization of LacY-LacZ biobrick (BBa_S04055)

Figure 1. Quantitative RT-PCR analysis of lacY and lacZ expression in E. coli cells. 
Expression of the lacZ and lacY genes was measured in control DH5α cells (BLUE) and BBa_S04055 recombinant DH5α cells (RED). Cells were cultured overnight in LB medium + antibiotic and total RNA harvested for qRT-PCR using 16S rRNA for normalization.

Quantitative real-time PCR

Quantitative real-time PCR analysis showed that both lacY and lacZ mRNA transcripts are expressed at high levels in recombinant E. coli cells (harboring the BBa_S04055 biobrick) as compared to control DH5α cells (Figure 1). Expression of lacY and lacZ is 9- fold and 2-fold higher, respectively, in recombinant cells with respect to 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.