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| | <partinfo>BBa_K185048 parameters</partinfo> | | <partinfo>BBa_K185048 parameters</partinfo> |
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| − | == Characterized by BNU-China 2019 ==
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| − | 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.
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| − | [[Image:2019_BNU-China_BBa_K185048_pic1.png| border | center | 400px]]<br>
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| − | In order to characterize 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, 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.
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| − | [[Image:2019_BNU-China_BBa_K185048_pic2.png| border | center | 400px]]<br>
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| − | <div class = "center">Figure 1 Relative population density at different temperatures</div>
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| − | With properties of relB, we can construct a relBE kill switch which can triggered under different conditions.
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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] 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.
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| | <h2> <b>Improved by BNU-China 2019</b> </h2> | | <h2> <b>Improved by BNU-China 2019</b> </h2> |
| | Please view <html><a href="https://parts.igem.org/Part:BBa_K3036008" target="_blank">BBa_K3036008</a> for more details<br> | | Please view <html><a href="https://parts.igem.org/Part:BBa_K3036008" target="_blank">BBa_K3036008</a> for more details<br> |
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.
