Difference between revisions of "Part:BBa K185048"

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	<b><font size="3">Properties</font></b>		<b><font size="3">Properties</font></b>
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	We improve the function of RelB by increasing its degradation rate so that once the expression stops, RelB degrades at a more rapid pace and leads to death of bacteria in a shorter period of time, which is a critical trait when assessing the validity of a biosafety system. To promote the degradation of RelB, we add a degradation-promoting tag RepA to RelB. RepA is a 16-amino-acid long peptide that conducts degradation of protein using native machinery in E. coli.		We improve the function of RelB by increasing its degradation rate so that once the expression stops, RelB degrades at a more rapid pace and leads to death of bacteria in a shorter period of time, which is a critical trait when assessing the validity of a biosafety system. To promote the degradation of RelB, we add a degradation-promoting tag RepA to RelB. RepA is a 16-amino-acid long peptide that conducts degradation of protein using native machinery in E. coli.
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	[[Image:2019 BNU-China BBa K3036008 pic1.png| border | center | 400px]]<br>		[[Image:2019 BNU-China BBa K3036008 pic1.png| border | center | 400px]]<br>
	<div class = "center">Fig. 1 Protein docking model for RelB-RelE interaction</div>		<div class = "center">Fig. 1 Protein docking model for RelB-RelE interaction</div>
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	In our improvement, RepA is placed at N-terminal of RelB. This strategy has two advantages: first, as is shown in Figure 1, the interaction with RelE is performed by the C-terminal domain of RelB, as a result, addition of degradation tag at N-terminal does not interfere with its function; second, adding the tag at N-terminal of RelB prevents the protein from losing the peptide through nonsense mutation.		In our improvement, RepA is placed at N-terminal of RelB. This strategy has two advantages: first, as is shown in Figure 1, the interaction with RelE is performed by the C-terminal domain of RelB, as a result, addition of degradation tag at N-terminal does not interfere with its function; second, adding the tag at N-terminal of RelB prevents the protein from losing the peptide through nonsense mutation.
	<br>		<br>
	<b><font size="3">Results</font></b>		<b><font size="3">Results</font></b>
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	We compare the function of RelB before and after improvement using the toxin-antitoxin system. As is shown in Figure 2, under same nonpermissive conditions, the bacteria containing the kill switch with improved RelB die at a notably higher rate than those with unimproved RelB, verifying the effect of the improvement. Otherwise, the OD600 shows no notable difference, indicating RelE kills bacteria without lysing the cell.		We compare the function of RelB before and after improvement using the toxin-antitoxin system. As is shown in Figure 2, under same nonpermissive conditions, the bacteria containing the kill switch with improved RelB die at a notably higher rate than those with unimproved RelB, verifying the effect of the improvement. Otherwise, the OD600 shows no notable difference, indicating RelE kills bacteria without lysing the cell.
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	[[Image:2019 BNU-China BBa K3036008 parts relB fig2.png | border | center | 800px]]<br>		[[Image:2019 BNU-China BBa K3036008 parts relB fig2.png | border | center | 800px]]<br>
	<div class = "center">Fig. 2 Difference between improved and unimproved RelB</div>		<div class = "center">Fig. 2 Difference between improved and unimproved RelB</div>
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	<b><font size="3">Experimental Approaches</font></b>		<b><font size="3">Experimental Approaches</font></b>
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	1.Transform kill switch systems with RelB or RepA-RelB into E. coli DH5alpha competent cells respectively. <br>		1.Transform kill switch systems with RelB or RepA-RelB into E. coli DH5alpha competent cells respectively. <br>
	2.Culture both strains with LB medium at 37℃ overnight. <br>		2.Culture both strains with LB medium at 37℃ overnight. <br>

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Revision as of 22:21, 21 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

Improved by BNU-China 2019

Please view BBa_K3036008 for more details

Properties
We improve the function of RelB by increasing its degradation rate so that once the expression stops, RelB degrades at a more rapid pace and leads to death of bacteria in a shorter period of time, which is a critical trait when assessing the validity of a biosafety system. To promote the degradation of RelB, we add a degradation-promoting tag RepA to RelB. RepA is a 16-amino-acid long peptide that conducts degradation of protein using native machinery in E. coli.
[[Image:2019 BNU-China BBa K3036008 pic1.png| border | center | 400px]]

In our improvement, RepA is placed at N-terminal of RelB. This strategy has two advantages: first, as is shown in Figure 1, the interaction with RelE is performed by the C-terminal domain of RelB, as a result, addition of degradation tag at N-terminal does not interfere with its function; second, adding the tag at N-terminal of RelB prevents the protein from losing the peptide through nonsense mutation.
Results
We compare the function of RelB before and after improvement using the toxin-antitoxin system. As is shown in Figure 2, under same nonpermissive conditions, the bacteria containing the kill switch with improved RelB die at a notably higher rate than those with unimproved RelB, verifying the effect of the improvement. Otherwise, the OD600 shows no notable difference, indicating RelE kills bacteria without lysing the cell.
[[Image:2019 BNU-China BBa K3036008 parts relB fig2.png | border | center | 800px]]

Experimental Approaches
1.Transform kill switch systems with RelB or RepA-RelB into E. coli DH5alpha competent cells respectively.
2.Culture both strains with LB medium at 37℃ overnight.
3.Equally divide each culture into two flasks. Put one of them at 37℃ and the other at 27℃.
4.Extract 5μl samples of each culture system every 6 hours. Diluted the samples 10^7 times and then spread them on solid LB-ampicillin (50 ng/µl) medium separately.
5.Count the number of colonies in 5 cm^2 per plate after cultured for 24 hours at 37℃.
6.Three replicas are tested in each group.