Difference between revisions of "Part:BBa K4061040"

Revision as of 02:09, 1 October 2021

eGFP antisense to downregulate eGFP (BBa_K4061002)

The mRNA antisense system has been known to downregulate gene expression at the translational level. The binding of the antisense mRNA to the coding mRNA will cause the degradation of the double-stranded mRNA by RNase H. Thus, the coding mRNA is unable to get translated into protein, resulting in a declining protein expression. This particular part, eGFP antisense was made to downregulate the expression of eGFP (BBa_K4061002). We designed the eGFP antisense such that the antisense mRNA fragment will bind with the first 22 nucleotides sequence of the eGFP, the RBS (BBa_B0034), and the last 10 nucleotides sequence of the OmpF promoter (BBa_K116500). Therefore, the binding of the antisense mRNA will inhibit the ribosome from binding to the RBS, which in turn inhibits the translation of the eGFP gene to protein.

Furthermore, our antisense eGFP construct is equipped with an Hfq binding site to stabilize the single strand mRNA fragment to protect it from degradation. The Hfq binding site that we utilize, named Spot 42, is an AU rich region and has a high specificity to Hfq protein. Hfq protein is endogenous to E.coli and it is known to interact with small antisense RNA that has AU rich region to modulate its stability and facilitate its binding to the target sequence.

In our context, the eGFP (BBa_K4061002) that we want to downregulate was controlled under an OmpC promoter (BBa_K116500) and RBS (BBa_B0034). Thus, the respective antisense strand would anneal to a region of the OmpC promoter and the RBS. For further utilization of this antisense fragment, it needs to be altered according to the promoter and the RBS used to control the eGFP. Sequence and Features