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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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| − | ===Usage and Biology===
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| − | <span class='h3bb'>Sequence and Features</span>
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| − | <partinfo>BBa_K185000 SequenceAndFeatures</partinfo>
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| − | ===Functional Parameters===
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| − | <partinfo>BBa_K185000 parameters</partinfo>
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| − | == Characterized by BNU-China 2019 ==
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| − | 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.
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| − | [[Image: 2019_BNU-China_BBa_K185048_pic1.png | border | center | 400px]]<br>
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| − | 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.
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| − | 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| border | center | 300px]]<br>
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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.
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| − | <b>Experimental approach</b>
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| − | 1. Transform the plasmids into E. coli DH5α competent cells.
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| − | 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;
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| − | 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;
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| − | 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;
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| − | 5. Count the number of colonies in 5 cm2 per plate after cultured for 24 hours at 37℃
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| − | 6. Three repicas are tested in each group.
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| − | <b>Reference</b>
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| − | [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.
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| | <!-- Add more about the biology of this part here | | <!-- Add more about the biology of this part here |
RelE is a toxin protein. And this part is composed with rbs BBa_B0030.
