Part:BBa_K3256007
Plac+YafQ*
This composite part contains lac promoter, RBS and YafQ*. More detail about the toxin is shown below.
Introduction
YafQ is a toxic gene of the toxin-antitoxin (TA) module in E. coli. YafQ protein will inhibit translation in E. coli. According to researches, gene expression in E. coli decreases 50% after 30 minutes and 70% after 90 minutes since YafQ starts expressing. DinJ is the cognate antitoxin gene of YafQ. DinJ protein will form complex with YafQ to deactivate YafQ. That is, proteins can be synthesized normally once DinJ is expressed. The modified amino acid sequence includes W30C(shown below). After these modifications, the toxin retains the function of suppressing the growth of E. coli, and makes the E. coli more stable from being killed easily.
Result
Cloning
We conducted colony PCR to verify that our target gene was correctly cloned into the E. coli BL21 (DE3).
Figure 1:Colony PCR result of toxin genes after cloning into E. coli BL21(DE3) YafQ* BBa_K3256442
Figure 1 was the electrophoresis results of the colony PCR with a marker on the left side and the target gene on the right side. The lengths are labeled beside each band. As a result, we successfully cloned five target genes, respectively, into E. coli.
Functional Test
After confirming the cloning of target genes, we tested their function by measuring the O.D. value after IPTG induction when the O.D. value reached 0.3 and compared with the O.D. value of the control. The O.D. values were documented in every five minutes for seven hours in total.
Figure 2 Growth curve of E. coli BL21(DE3) with 500uM IPTG induction YafQ toxin gene (blue), and control (orange).</p>
Calculating Toxicity of Toxin Genes
We first fit the control’s experiment data to the following equation, dBT/dt=g⋅BT(1−BT/BMax), and we fit the induced data to another equation, dBT/dt=g⋅BT(1−BT/BMax)−Ttoxin⋅BN⋅[toxin]. Next, we compared the two equations to calculate Ttoxin, the toxicity of the toxin gene (Figure 3). In the end, we chose the ydfD gene, which had the most significant toxicity, and the ccdB gene, which had relatively weak toxicity, moving on to the Functional test with mutagens.
Figure 3:Toxicity of different toxin genes. Ttoxin: The toxicity of toxin gene.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 358
- 1000COMPATIBLE WITH RFC[1000]
References
Prysak, M. H., et al. (2009). "Bacterial toxin YafQ is an endoribonuclease that associates with the ribosome and blocks translation elongation through sequence‐specific and frame‐dependent mRNA cleavage." Molecular Microbiology 71(5): 1071-1087.
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