Difference between revisions of "Part:BBa K2145100"

Latest revision as of 23:59, 30 October 2016

GFP/RFP reporter (Forward/Forward) separated by three dCas clamp sites

This part contains two fluorescent protein expression units (for RFP and GFP) with promoters, RBSs, CDSs, and terminators, both facing backward, separated by a 282-bp spacer containing three gRNA binding sites for dCas. Parts were amplified from iGEM Registry (BBa_J04450 (RFP) and BBa_I13522 (GFP)) and assembled with golden gate assembly to make the full part. The reporter is designed to demonstrate the effect, if any, of binding a dCas "clamp" between two genes (the idea being that such a clamp can limit propagation of supercoiling between the genes, functionally isolating them). Direction of GFP: Forward, toward the clamps; Direction of RFP: Forward, away from the clamps

This part is one of a 4-plasmid set (K2145100, K2145101, K2145102, and K2145103). Other plasmids in this set are similar, but have GFP and RFP expression cassettes pointing in different directions. Different arrangement and orientations are expected to generate different patterns of supercoiling, which translates to different patterns of expression in RFP and GFP if supercoiling creates interference between the two. Addition of dCas and gRNAs matching the clamp sites are expected to reduce supercoiling, leading to more similar expression between the plasmids in this set.

Sequence and Features: Sequencing confirmed the presence of both GFP and RFP.