Difference between revisions of "Part:BBa K185047"
Line 18: Line 18:
 
== Characterized by BNU-China 2019 ==
 
== 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.
+
We characterize relE (BBa_K185000) by an antitoxin-toxin system, in which the downstream relE gene encoding for a stable toxin is constantly expressed, and the upstream relB (BBa_K185048) gene encodes for a labile antitoxin under the control of a temperature-sensitive RNA thermometer (BBa_K115002). Without counteracted by antitoxin RelB, RelE shuts down protein synthesis and causes the death of microbe by cleaving mRNA within the ribosomal A site [1]. In addition, the RNA thermometer allows expression of RelB at 37℃, but inhibits expression at 27℃, which leads to excess expression of RelE. As a result, we can characterize relE in a cell density-dependent manner in Escherichia coli K-12.
  
−
[[Image:2019_BNU-China_BBa_K185048_pic1.png| border | center | 400px]]<br>
+
[[Image: 2019_BNU-China_BBa_K185048_pic1.png | border | center | 400px]]<br>
  
−
In order to characterize relB, we take E. coli introduced with a vector with the same backbone as control group.  
+
In order to characterize the toxicity of protein encoded by relE and whether it can be inhibited by antitoxin 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.  
+
As is shown in Fig.1, there is nearly no difference of the relative population density between control and experimental groups at 37℃, which indicates RelE interacts with RelB and thereby the cleavage of mRNA by RelE is inhibited. However, the population density of experimental group shows a significant decrease compared to control group at 27℃, which indicates the protein encoded by relE is lethal to E. coli.
 +
 
 +
[[Image:2019_BNU-China_BBa_K185048_pic2.png| border | center | 300px]]<br>
  
−
[[Image:2019_BNU-China_BBa_K185048_pic2.png| border | center | 400px]]<br>
 
 
<div class = "center">Figure 1 Relative population density at different temperatures</div>
 
<div class = "center">Figure 1 Relative population density at different temperatures</div>
  
−
With properties of relB, we can construct a relBE kill switch which can triggered under different conditions.
+
With properties of relE, we can construct a kill switch in engineered microbe to ensure lab safety.  
  
 
<b>Experimental approach</b>
 
<b>Experimental approach</b>
Line 42: Line 43:
 
<b>Reference</b>
 
<b>Reference</b>
  
−
[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.
+
[1] Overgaard M , Borch J , Gerdes K . RelB and RelE of Escherichia coli Form a Tight Complex That Represses Transcription via the Ribbon–Helix–Helix Motif in RelB[J]. Journal of Molecular Biology, 2009, 394(2):0-196.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 16:32, 21 October 2019

RelE toxin

The relE toxin is an RNase that preferentially cleaves mRNAs bound to the ribosome at the second position of stop codons. Stop codons not only signal the end of the protein coding sequence but also serve as the binding site for release factors, which promote release of the nascent polypeptide and facilitate recycling of ribosomes for further rounds of translation. Thus truncated mRNA by cleavage of relE lacks appropriate termination signals, which causes the accumulation of stalled ribosomes and these mRNAs are unable to promote release factor binding, nascent polypeptide release, and ribosome recycling. As a result, expression of the relE gene has been shown to severely inhibit translation and prevent colony formation. RelE display codon-specific cleavage of mRNAs in the ribosomal A site, that is to say, among stop codons UAG is cleaved with fast, UAA intermediate and UGA slow rate(UAG>UAA>UGA).

In our design, we add a his-tag at the end of RelE sequence to detect the expression of relE protein. Then we mutated the stop condon from UGA to UAA to make relE toxin inhibit its own traslation moderately.Most importantly, you can get this part in BBa_K185000 or BBa_K185004


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterized by BNU-China 2019

We characterize relE (BBa_K185000) by an antitoxin-toxin system, in which the downstream relE gene encoding for a stable toxin is constantly expressed, and the upstream relB (BBa_K185048) gene encodes for a labile antitoxin under the control of a temperature-sensitive RNA thermometer (BBa_K115002). Without counteracted by antitoxin RelB, RelE shuts down protein synthesis and causes the death of microbe by cleaving mRNA within the ribosomal A site [1]. In addition, the RNA thermometer allows expression of RelB at 37℃, but inhibits expression at 27℃, which leads to excess expression of RelE. As a result, we can characterize relE in a cell density-dependent manner in Escherichia coli K-12.

2019 BNU-China BBa K185048 pic1.png

In order to characterize the toxicity of protein encoded by relE and whether it can be inhibited by antitoxin RelB, we take E. coli introduced with a vector with the same backbone as control group.

As is shown in Fig.1, there is nearly no difference of the relative population density between control and experimental groups at 37℃, which indicates RelE interacts with RelB and thereby the cleavage of mRNA by RelE is inhibited. However, the population density of experimental group shows a significant decrease compared to control group at 27℃, which indicates the protein encoded by relE is lethal to E. coli.

2019 BNU-China BBa K185048 pic2.png

Figure 1 Relative population density at different temperatures

With properties of relE, we can construct a kill switch in engineered microbe to ensure lab safety.

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] Overgaard M , Borch J , Gerdes K . RelB and RelE of Escherichia coli Form a Tight Complex That Represses Transcription via the Ribbon–Helix–Helix Motif in RelB[J]. Journal of Molecular Biology, 2009, 394(2):0-196.

Sequence and Features

BBa_K185047 SequenceAndFeatures