n our project, we aimed to produce polyPphosphate（polyP） in vitro and increase its production
by engineering a certain “bacteria factory” through synthetic biology methods.
The biosynthesized polyP could clinically help improve intestinal health.
PART1: New Application of polyP
After realizing that inflammatory bowel disease (IBD) is a serious problem all over the world,
we exchanged views with Prof. Wenjie Guo, an associated professor of Nanjing University School of Life Sciences,
who had done a number of research in IBD.
He told us that according to his previous studies, long-chain polyP might be promising for treating IBD.
PolyP is a kind of inorganic polymer existing widely in both eukaryotes and prokaryotes.
However, during our following investigation, we discovered
that it was difficult to obtain ideal long-chain polyP through the currently prevalent chemical synthesis pathway,
which limited its mass production for potential clinical use.
Chemical synthesis process requires high temperature and high pressure,
which not only cost a lot but was also not eco-friendly.
Compared to widely-existing biosynthesis pathway in vivo, polyP produced through chemical methods
had shorter chain-length and were more unstable in nature.
Therefore, in our project, we employ E. coli’s biosynthesis pathway of polyP to synthesize polyP
from phosphorus wastewater and increase the production of polyP by inserting extra genes, vgb and maze, into E. coli genome.
The increase of production is the major innovation point of our project.
PART2: Reconstruct bacteria to produce polyP in vitro
In order to biosynthesize polyP, we planned to construct a recombinant plasmid containing polyP synthetase of bacteria,
and transform it into E. coli BL21. When it comes to choosing the optimal polyP synthetase,
our modeling compared two candidate enzymes, PPK1, which is more common among bacteria,
and PPK2B, which exists in C. glutamicum, by simulating the binding
between the mentioned two enzymes and polyP with different lengths.
We found that with a lower binding energy, the binding between polyP and PPK1 is tighter than
that between same-length polyP and PPK2B, supporting the conclusion to choose PPK1 as polyP synthetase.
Besides, to explore the effects of PPK1 on E. coli itself,
we measured the growth curves of both modified and unmodified strains
by means of quantitatively describing the content of polyP and the abundance of bacteria.
It is also worth mentioning that in view of the significance of protecting the environment,
we used synthetic wastewater containing phosphorus to culture bacteria.
As a result, through the “bacteria factory”, we could obtain a large amount of polyP,
and could use TBE-Urea PAGE assay to estimate the range of molecular weight of polyP,
calculating the polymerization degree of polyP to ensure we got the chain length we wanted.
The fitting of the first experiment was good in the early stage,
but there was no obvious steady state in the late stage in synthetic medium.
Confused, we exchanged views with our partner team NJMU-China,
they suggested we prolong culture time and reduce the interval of measurement time.
Taking their advice, we gained curve of OD600 and OD700 with a much smaller fluctuation,
which helps us clearly estimate the number of bacteria and the concentration of phosphate in synthetic medium.
PART3: Increase of polyP production
After successfully biosynthesizing polyP, we wondered if the yield was up to standard for medicinal use,
so we interviewed professor Ding. He spoke highly of our project but pointed out
that the productivity of our engineered bacteria had yet to be improved.
After consulting several papers, we planned to use whole EPVM (including ppkI, mazE and vgb)
to help the bacteria produce more polyP and grow better.
Apart from ppk1, the expression of both vgb and mazE can help increase the biomass of bacteria and produce more polyP.
Compared with the first experiment, the OD700 of EPVM decreased greatly,
which demonstrated the increase in our polyP yield.
With the yield of polyP living up to our expectations, the improvement of our parts was declared a success.