Difference between revisions of "Part:BBa K185048"
 
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== Characterized by BNU-China 2019 ==
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<h2> <b>Improved by BNU-China 2019</b> </h2>
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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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<br>
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<b><font size="3">Properties</font></b>
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<br>
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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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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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<die align="center"><img src="https://static.igem.org/mediawiki/parts/b/b6/2019_BNU-China_BBa_K3036008_pic1.png"></div>
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<div class = "center">Fig. 1 Protein docking model for RelB-RelE interaction</div>
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<br>
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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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<br>
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<b><font size="3">Results</font></b>
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<br>
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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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<br>
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<die align="center"><img src="https://static.igem.org/mediawiki/parts/b/b8/2019_BNU-China_BBa_K3036008_parts_relB_fig2.png" width="900px" height="250px"/></div>
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<div class = "center">Fig. 2 Difference between improved and unimproved RelB</div>
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<br>
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<b><font size="3">Experimental Approaches</font></b>
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<br>
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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>
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<br>
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[[Image:2019_BNU-China_BBa_K185048_pic1.png| border | center | 400px]]<br>
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<h2> <b>Attention （Added by 2020 Fudan)</b> </h2>
  
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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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<b><font size="3">Homonymic “Relb”</font></b>
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<br>
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When searching for bacterial RelB toxin, we accidentally found another homonymic “Relb” that sometimes also documented as “RelB” but in vertebrate. Relb is a transcription factor of nuclear factror-kappa B (NF-κB) family and plays a crucial role in inducing inflammation, autoimmune diseases, multiple sclerosis, etc. iGEM teams who want to utilize RelB for Toxin/antitoxin system should distinguish it for the other. Relb for vertebrate seems to haven’t been used by iGEM team before [1].<br>
  
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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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<b><font size="3">Chromosomally encoded RelE/RelB in gut microbiota</font></b>
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<br>
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[[Image:2019_BNU-China_BBa_K185048_pic2.png| border | center | 400px]]<br>
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What’s more, RelE/RelB and other toxin/antitoxin modules have homology searches on bacterial chromosomes. RelB-RelE complex can inhibit the promotor of chromosomally encoded RelE/RelB [2].<br>
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<div class = "center">Figure 1 Relative population density at different temperatures</div>
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<br>
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This new information learned from literature that need attention was added by <html><a href="https://2020.igem.org/Team:Fudan" target="_blank">2020 Fudan team</a>.<br>
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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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<br>
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<b>Experimental approach</b>
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<b><font size="3">Reference</font></b>
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<br>
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1. Transform the plasmids into E. coli DH5α competent cells.
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[1] Yang, Meng-Ge et al. “Biological characteristics of transcription factor RelB in different immune cell types: implications for the treatment of multiple sclerosis.” Molecular brain vol. 12,1 115. 27 Dec. 2019, doi:10.1186/s13041-019-0532-6<br>
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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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[2] Unterholzner, Simon J et al. “Toxin-antitoxin systems: Biology, identification, and application.” Mobile genetic elements vol. 3,5 (2013): e26219. doi:10.4161/mge.26219<br>
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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>
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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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Latest revision as of 13:31, 27 October 2020

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.

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.
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.


Attention （Added by 2020 Fudan)

Homonymic “Relb”
When searching for bacterial RelB toxin, we accidentally found another homonymic “Relb” that sometimes also documented as “RelB” but in vertebrate. Relb is a transcription factor of nuclear factror-kappa B (NF-κB) family and plays a crucial role in inducing inflammation, autoimmune diseases, multiple sclerosis, etc. iGEM teams who want to utilize RelB for Toxin/antitoxin system should distinguish it for the other. Relb for vertebrate seems to haven’t been used by iGEM team before [1].
Chromosomally encoded RelE/RelB in gut microbiota
What’s more, RelE/RelB and other toxin/antitoxin modules have homology searches on bacterial chromosomes. RelB-RelE complex can inhibit the promotor of chromosomally encoded RelE/RelB [2].

This new information learned from literature that need attention was added by 2020 Fudan team.

Reference
[1] Yang, Meng-Ge et al. “Biological characteristics of transcription factor RelB in different immune cell types: implications for the treatment of multiple sclerosis.” Molecular brain vol. 12,1 115. 27 Dec. 2019, doi:10.1186/s13041-019-0532-6
[2] Unterholzner, Simon J et al. “Toxin-antitoxin systems: Biology, identification, and application.” Mobile genetic elements vol. 3,5 (2013): e26219. doi:10.4161/mge.26219