Team:JiangnanU China/Description

JiangNan

Why Do We Choose This Project?

Back to Febuary, 2019, when we were doing an experiment, our bacteria were all killed by bacteriophage T4. 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 to our major, phage infection can cause a loss of $100,000 if this happens in a 500-ton fermenter. 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 this problem.

Bacteriophage, also called phage or bacterial virus, any of a group of viruses that infect bacteria. Bacteriophages were discovered independently by Frederick W. Twort in Great Britain (1915) and Félix d’Hérelle in France (1917). D’Hérelle coined the term bacteriophage, meaning “bacteria eater”, to describe the agent’s bactericidal ability. Bacteriophages also infect the single-celled prokaryotic organisms known as archaea.[1]


Phage T4 is a virulent phage，it uses the metabolic machinery of the host cell to produce progeny viruses and kill the host in the process. Depending upon the phage, the nucleic acid can be either DNA or RNA but not both. Phage T4 is a double-stranded DNA virus. Phages can reproduce on its own. So they need a host body to do their gene replication. And T4 has got a host named E. coli bacterial which is known as the colon bacteria. Bacteriophage T4 is the most well-studied member of Myoviridae, the most complex family of tailed phages. T4 assembly is divided into three independent pathways: the head, the tail and the long tail fibers. Six long tail fibers are attached to the baseplate’s periphery and are the host cell’s recognition sensors. The sheath and the baseplate undergo large conformational changes during infection.

For us human, we get vaccinated in order to avoid being infected by virus. So, we thought about introducing a protein gene into the E. coli BL21, enabling it to protect itself from the phage attack.
If we can successfully construct a gene circuit where we connect anti-protein gene with promoters and control the expression, we can greatly reduce the possibility of Escherichia coli BL21 being killed during the experiment and help the factories 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 bacteriophage T4 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[2], which are both anti-phage genes that can show resistance to the attack from the bacteriophage T4.

What Do We Do?

We connect anti-protein with an inducible promoter PputA. For fear that the anti-protein could not work successfully, we connect kill switch[3] with the inducible promoter PglcF, to avoid the replication and release of phages. Before that, we have proved that the inducible promoter PputA and PglcF 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 four 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 advised, our strain should grow as robust as the original strain. Otherwise, it could not be applied under current fermentation condition, so we applied a Gray Relation Analysis[5] model with EWM weights[4] and weights advised by experts, to analyse the correlation of growth curve between our strains and original strains 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[6], which allows us to choose or design promoters with desired strength in our circuit without extra experiments.

References[1-6]

[1] Yap ML, Rossmann MG. Structure and function of bacteriophage T4. Future Microbiol.2014;9(12):1319–1327. doi:10.2217/fmb.14.91
[2] 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.
[3] 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.
[4]LI X, WANG K, LIU L, et al. Application of the Entropy Weight and TOPSIS Method in Safety Evaluation of Coal Mines [J]. Procedia Engineering, 2011, 26(4): 2085-2091.
[5] KUO Y, YANG T, HUANG G W. The use of gray relational analysis in solving multiple attribute decision-making problems [J]. Computers & Industrial Engineering, 2008, 55(1): 80-93.
[6] 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.
back