Team:JiangnanU China/Description
Why we chose 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.
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 Escherichia coli BL21, enabling it to protect itself from the phage attack.
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 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, 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 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 model with EWM weights 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, which allows us to choose or design promoters with desired strength in our circuit without extra experiments.
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 model with EWM weights 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, which allows us to choose or design promoters 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. Yap ML, Rossmann MG. Structure and function of bacteriophage T4. Future Microbiol. 2014;9(12):1319–1327. doi:10.2217/fmb.14.91
10. 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.
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. Yap ML, Rossmann MG. Structure and function of bacteriophage T4. Future Microbiol. 2014;9(12):1319–1327. doi:10.2217/fmb.14.91
10. 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.
