Difference between revisions of "Part:BBa K185048"
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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>
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<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.
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[[Image:2019 BNU-China BBa K3036008 pic1.png| border | center | 400px]]<br>
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<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.
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<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. 
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[[Image:2019 BNU-China BBa K3036008 parts relB fig2.png | border | center | 800px]]<br>
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<div class = "center">Fig. 2 Difference between improved and unimproved RelB</div>
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<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>
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2.Culture both strains with LB medium at 37℃ overnight. <br>
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3.Equally divide each culture into two flasks. Put one of them at 37℃ and the other at 27℃. <br>
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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. <br>
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5.Count the number of colonies in 5 cm^2 per plate after cultured for 24 hours at 37℃.<br>
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6.Three replicas are tested in each group. <br>

Revision as of 22:14, 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 103
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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]]

Fig. 1 Protein docking model for RelB-RelE interaction
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]]
Fig. 2 Difference between improved and unimproved RelB
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.