Difference between revisions of "Team:Fudan-TSI/Results/Reverse Transcription"

RT is expressed under an IPTG controlled promoter, we constructed a series of promoters by placing a LacO fragment under a stable promoter, and hopes to determine under which we could achieve most stringent control. The constructs we tested are—T5, T5-LacO, T7-LacO, LacUV5-LacO, J23119-LacO.

We initially attempted to verify RT’s expression through an EGFP fusion protein. We fused the EGFP to the C’ end of pol protein, linked by a GS tag. But this construct proved unsuccessful (data not shown), possibly due to the length and complexity of the gag-pol polyprotein. Then we turned to directly expressing EGFP in the place of RT under the control of IPTG. If EGFP can be successfully expressed, so should RT. And results are obtained for each induction promoter construct (figure would be supplemented in presentation and poster).

We found through careful examination that this failure is due to problems with our plasmid construct, so we moved the RT to another tested plasmid, and through SDS-PAGE, verified its successful expression (Fig. 3).