Difference between revisions of "Part:BBa K185000"

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	RelE is a toxin protein. And this part is composed with rbs BBa_B0030.		RelE is a toxin protein. And this part is composed with rbs BBa_B0030.
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		+	<!-- Add more about the biology of this part here
		+	===Usage and Biology===
		+
		+	<!-- -->
		+	<span class='h3bb'>Sequence and Features</span>
		+	<partinfo>BBa_K185000 SequenceAndFeatures</partinfo>
		+
		+
		+	<!-- Uncomment this to enable Functional Parameter display
		+	===Functional Parameters===
		+	<partinfo>BBa_K185000 parameters</partinfo>
		+	<!-- -->
		+
		+	== 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.
		+
		+	[[Image: 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.
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		+	[[Image:2019_BNU-China_BBa_K185048_pic2.png]]
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		+	<div class = "center">Figure 1 Relative population density at different temperatures</div>
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		+	With properties of relE, we can construct a kill switch in engineered microbe to ensure lab safety.
		+
		+	<b>Experimental approach</b>
		+
		+	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.
		+
		+	<b>Reference</b>
		+
		+	[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

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Revision as of 10:08, 9 October 2019

RelE toxin+Rbs30

RelE is a toxin protein. And this part is composed with rbs BBa_B0030.

Sequence and Features

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.

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.

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