Revision as of 03:48, 22 October 2019

Ready to use mRFP1-CarH device (strong)

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 weak Anderson promoter (BBa_J23114) 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 ^[1]. When excited by green light, the photosensitive Co-C bond breaks ^[2], 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

System Responsiveness

An experiment was designed to see, if the CarH sysem is modular (protocol). An overnight culture (without cobalamin) was diluted with liquid LB media (containing 10 μM cobalamin), thus allowing the CarH protein to repress in the dark state. The bacteria were kept varying amounts on time in the dark and later brought to light and we were able to observe a change in fluorescence divided by OD 600. The bacteria were grown in 30°C.

<b>Figure 1.</b>Figure 2. An examplary graph showing how the fluorescence divided by OD 600 changes in the same bacteria containing tube when it is brought from the light to the dark and then again to the dark. The last state the bacteria were in before measuring is denoted as the colour of the point, dark blue point indicates the dark and the light orange poitn denotes the light. TG1 E.coli strain grown in 30°C

Full experimental protocol can be found here

References

1. ↑ Padmanabhan S, Pérez-Castaño R, Elías-Arnanz M. B12-based photoreceptors: from structure and function to applications in optogenetics and synthetic biology. Current Opinion in Structural Biology. 2019;57:47-55. doi:10.1016/j.sbi.2019.01.020
2. ↑ Chatelle C, Ochoa-Fernandez R, Engesser R, et al. A Green-Light-Responsive System for the Control of Transgene Expression in Mammalian and Plant Cells. ACS Synth Biol. 2018;7(5):1349-1358. doi:10.1021/acssynbio.7b00450

Sequence and Features