Team:JiangnanU China/Description

Project Description

Why we chose this project? Back to Febuary,2019, when we were doing an experiment, our bacteria were all killed by the T4 phages. That was a really awful experience because our whole experiment plan was interrupted. As we all know, phage infection is a common phenomenon in the lab, which will delay the experimental process of the whole lab. In the fermentation industry, which is closely related with our major, phage infection can cause a loss of $100,000 if this happens in a 500 ton ferment tank. We checked the previous iGEM projects about phages but didn’t find a project about protecting bacteria from phage attack. Therefore, we started to brainstorm, trying to solve such problem.

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For us human, , we get vaccinated in order to avoid being infected by virus. So, we thought about introducing a protein gene into the Escherichia coli BL21, enabling it to protect itself from the phage attack.

If we can successfully construct this gene circuit and make it work, we can greatly reduce the possibility of Escherichia coli BL21 being killed during the experiment as well as help the factories to save money and time. To conclude, we build this gene circuit to reduce the mortality of Escherichia coli BL21 to help the biological researchers and factories.

What is The Main Problem?

As phages have a high rate of mutant and a high degree of specificity, it is hard to protect the Escherichia coli BL21 from all kinds of phage attack. So, we can only choose T4 phages which killed our bacteria before and reduce its impact on the Escherichia coli BL21 . Besides, there is not so much literature focusing on the anti-phage mechanism of Escherichia coli BL21. We try to protect the Escherichia coli BL21 from the gene level. And luckily, according to the reference, we have found abpA and abpB, which are both anti-phage genes that can show resistance to the attack from the T4 phages.

What Do We Do?

We connect anti-protein with an inducible promoter putA. For fear that the anti-protein could not work successfully, we connect kill switch with the inducible promoter glcF, to avoid the replication and release of phages. Before that, we have proved that the inducible promoter putA and glcF can work efficiently by using report gene gfp and rfp. According to the reference, we have found anti-phage gene abpA and abpB, but they didn’t work well. We introduced phages into the medium and let them attack phages. Finally , we found six anti-phage genes according to its mutant sites and connect gntR with abpA and abpB. To our surprise, the gene circuit shows great resistance to the phage attack. Meanwhile, as the stakeholders adviced, our strain should grow as robust as the original strain or it could not be applied under current fermentation condition, so we applied an Gray Relation Analysis model with EWM weights and weights advised by experts, to analyse the correlation of growth curve between our strains and original strain in order to select the most suitable strain. Besides, due to the potential problems such as the promoter leakage and inclusion body ,we developed a quantitative design method for phage-induced promoters based on strength prediction using artificial neural network, which allows us to choose or design promoter with desired strength in our circuit without extra experiments.

References[1-9]

1. Lam KL, Ishitsuka Y, Cheng Y, Chien K, Waring AJ, Lehrer RI, Lee KY: Mechanism of supported membrane disruption by antimicrobial peptide protegrin-1. J Phys Chem B 2006, 110(42):21282-21286.
2. Liebig HD, Ruger W: Bacteriophage T4 early promoter regions. Consensus sequences of promoters and ribosome-binding sites. J Mol Biol 1989, 208(4):517-536.
3. Meng H, Wang J, Xiong Z, Xu F, Zhao G, Wang Y: Quantitative design of regulatory elements based on high-precision strength prediction using artificial neural network. PLoS One 2013, 8(4):e60288.
4. Ohlendorf R, Vidavski RR, Eldar A, Moffat K, Moglich A: From dusk till dawn: one-plasmid systems for light-regulated gene expression. J Mol Biol 2012, 416(4):534-542.
5. Wilkens K, Ruger W: Characterization of bacteriophage T4 early promoters in vivo with a new promoter probe vector. Plasmid 1996, 35(2):108-120.
6. Yasui R, Washizaki A, Furihata Y, Yonesaki T, Otsuka Y: AbpA and AbpB provide anti-phage activity in Escherichia coli. Genes Genet Syst 2014, 89(2):51-60.
7. Zhang C, Qin J, Dai Y, Mu W, Zhang T: Atmospheric and room temperature plasma (ARTP) mutagenesis enables xylitol over-production with yeast Candida tropicalis. J Biotechnol 2019, 296:7-13.
8. Zhang X, Zhang XF, Li HP, Wang LY, Zhang C, Xing XH, Bao CY: Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool. Appl Microbiol Biotechnol 2014, 98(12):5387-5396.
9. 崔晓莉: 铜绿假单胞菌应答多株噬菌体感染相关基因的筛选及噬菌体C11基因组的功能注释. 硕士. 天津科技大学; 2016.