Difference between revisions of "Part:BBa K185048"

Revision as of 11:32, 16 October 2019

RelB antitoxin

RelB antitoxin, which can form a heterotetrameric (relB-relE)2 structure when binding with relE,can restore the E.coli growth.The heterotetrameric (relB-relE)2 structure is too large to fit into the A site, so the toxic relE can be neutralized. Overexpression of relB restored protein synthesis and colony formation.

Sequence and Features

Characterized by BNU-China 2019

The antitoxin encoded by relB (BBa_K185048), binds and inhibits RelE from shutting down protein synthesis and causing the death of microbe by cleaving mRNA [1]. Hence, we characterize relB (BBa_K185048) by an antitoxin-toxin system, in which the downstream relE (BBa_K185000) gene encodes for a stable toxin, and the upstream relB gene encodes for a labile antitoxin under the control of a temperature-sensitive RNA thermometer (BBa_K115002). In addition, the RNA thermometer allows expression of relB at 37℃, but it inhibits translation at 27℃, which leads to excess of relE. As a result, we can characterize relB in a cell density-dependent manner in Escherichia coli K-12.

In order to characterize relB, we take E. coli introduced with a vector with the same backbone as control group.

As is shown in Fig.1, the population density of experimental group shows a significant decrease compared to control group at 27℃, which indicates RelE could induce the death of microbe successfully. However, there is nearly no difference of the relative population density between control and experimental groups at 37℃, which indicates RelB counteracts RelE and thereby inhibits the cleavage of mRNA by RelE.

With properties of relB, we can construct a relBE kill switch which can triggered under different conditions.

Experimental approach

1. Transform the plasmids into E. coli DH5α competent cells. 2. A strain containing a vector with same backbone is used as control. Experimental groups and control groups are both cultured in 60mL LB-ampicillin (50 ng/µl) medium overnight at 37℃, 200rpm; 3. Equally divide each group into two flasks, which is 30mL respectively. One of each group is cultured at 27℃, 200rpm and the other at 37℃, 200rpm; 4. Extract 5μl samples of each culture system every 6 hours. Diluted all of the samples to 107 times and then spread them on solid LB-ampicillin (50 ng/µl) medium separately; 5. Count the number of colonies in 5 cm2 per plate after cultured for 24 hours at 37℃ 6. Three repicas are tested in each group.

Reference

[1] Andreas Bøggild, Sofos N, Andersen K R, et al. The Crystal Structure of the Intact E. coli ReIBE Toxin-Antitoxin Complex Provides the Structural Basis for Conditional Cooperativity[J]. Structure, 2012, 20(10):1641-1648.

Sequence and Features