Difference between revisions of "Part:BBa K185048"
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5.Count the number of colonies in 5 cm^2 per plate after cultured for 24 hours at 37℃.<br>  
 
5.Count the number of colonies in 5 cm^2 per plate after cultured for 24 hours at 37℃.<br>  
 
6.Three replicas are tested in each group. <br>
 
6.Three replicas are tested in each group. <br>
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 +
===Attention by 2020 Fudan===
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=====1. Potential pathogenicity of RelE =====
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Although, RelE/RelB module poses minimized adverse effects on hosts as it expanding in microbial communities <ref>Jones, Brian V. “The human gut mobile metagenome: a metazoan perspective.” Gut microbes vol. 1,6 (2010): 415-31. doi:10.4161/gmic.1.6.14087</ref>, it has several other effects on human intestine. Firstly, RelE is a very rare toxin that has the activity against both prokaryotic and eukaryotic organisms in a similar mechanism. It has been observed that RelE can induce apoptosis in cultured human cell lines. Secondly, RelE/RelB module tends to have unexpected enrichment in bacteria that is not observed for other toxin/antitoxin modules such as MazE/MazF and ParD/ParE. Such enrichment may be associated with the formation of persistence of microbes in the gut. Thirdly, the divisions of bacteria carrying homologues of the RelE toxin involved in comprising the human gut microbiota.
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All this effect only been testified in artificial mammalian gene expression systems, so whether it has tangible impact on the human gut microbiome need further study. iGEM teams that focus on intestinal probiotics and have special safety requirements ought to pay special attention to these.
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=====2. Chromosomally encoded RelE/RelB in gut microbiota=====
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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<ref>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</ref>.
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=====3. Homonymic “Relb”=====
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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<ref>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</ref>.
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This new information learned from literature that need attention was added by 2020 Fudan team.

Revision as of 07:21, 22 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 by 2020 Fudan=== =====1. Potential pathogenicity of RelE ===== Although, RelE/RelB module poses minimized adverse effects on hosts as it expanding in microbial communities Jones, Brian V. “The human gut mobile metagenome: a metazoan perspective.” Gut microbes vol. 1,6 (2010): 415-31. doi:10.4161/gmic.1.6.14087, it has several other effects on human intestine. Firstly, RelE is a very rare toxin that has the activity against both prokaryotic and eukaryotic organisms in a similar mechanism. It has been observed that RelE can induce apoptosis in cultured human cell lines. Secondly, RelE/RelB module tends to have unexpected enrichment in bacteria that is not observed for other toxin/antitoxin modules such as MazE/MazF and ParD/ParE. Such enrichment may be associated with the formation of persistence of microbes in the gut. Thirdly, the divisions of bacteria carrying homologues of the RelE toxin involved in comprising the human gut microbiota. All this effect only been testified in artificial mammalian gene expression systems, so whether it has tangible impact on the human gut microbiome need further study. iGEM teams that focus on intestinal probiotics and have special safety requirements ought to pay special attention to these. =====2. 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/RelBUnterholzner, Simon J et al. “Toxin-antitoxin systems: Biology, identification, and application.” Mobile genetic elements vol. 3,5 (2013): e26219. doi:10.4161/mge.26219. =====3. 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 beforeYang, 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. This new information learned from literature that need attention was added by 2020 Fudan team.