Part:BBa_K3032074

Ready to use mRFP1-CarH device (weak)

Introduction

The CarH system

This is a ready to use device based on the combination of BBa_K3032070 and BBa_K3032073 parts, where BBa_K3032070 part is a mRFP1 reporter under a light-inducible CarH promoter and a strong RBS (BBa_B0034) and BBa_K3032073 part is a CarH repressor under a strong Anderson promoter (BBa_J23110) and strong RBS (BBa_B0034).

CarH protein from Thermus thermophilus incorporates cobalamine (Vitamin B12), which has a photosensitive Co-C bond, as a prosthetic group. In the dark, CarH is a tetramer that binds strongly to DNA and represses protein translation . When excited by green light, the photosensitive Co-C bond breaks , this disrupts the tetramer structure, and then gene expression can start.

An animation showing the CarH system mechanism of action.

Results

Experimental procedure

For our experimental procedure, we constructed a plasmid carrying a CarH cassette under a constitutive Anderson promoter (Part:BBa_J23114), and an RFP cassette under the CarH inducible promoter of our design. The experiment was conducted with 20 μM cyanocobalamin, the cells were grown in 30 degrees celsius overnight. With CarH gene under a weak promoter, we were able to observe a 45 fold difference between bacteria grown in light and in the dark

<b>Figure 1.</b> Figure 1. A graph showing the difference in mRFP expression between CarH device containing E. coli grown in the dark and in the light. This graph is for both Part:BBa_K3032074, mRFP1-CarH device (strong), and mRFP1-CarH device (weak) Part:BBa_K3032074, Bacteria were grown in 30 °C for 14 hours, TG1 strain