PART1: NEW DOCUMENTATION TO THE EXISTING PARTS
We contributed to the iGEM part library by adding the information to two part pages [Part: BBa_K3022002] and [Part: BBa_K1807009].
1、New documentation to CFppk1 gene:
We made some contributions to the expression and purification of CFppk1 gene, that is,
we took ppk1 in Citrobacter freundii ATCC 8090 as a comparison and validated its activity through the experiment.
We found that like in Citrobacter freundii ATCC 8090, ppk1 can also produce polyP in the E.coli, which proved our hypothesis.
We supplied experimental conditions and our results to CFppk1 gene to validate its function in E.coli BL21.
2、New documentation to ECppk1 gene:
We made some contributions to ECppk1, which is ppk1 in E.coli BL21, from literature review.
We elaborated the mechanism of its function and its purification method.
What’s more, according to the literature, measurements of its efficiency in producing polyP
are also supplied into the existing part.
PART2: IMPROVEMENT OF EXISTING PARTS AND CONSTRUCTION OF NEW PARTS
We created a new part [Part: BBa_K3731003] in order to improve the existing part [Part: BBa_K1807009].
And we uploaded three new parts [Part: BBa_K3731001], [Part: BBa_K3731002] and [Part: BBa_K3731007] based on our experiment.
1. The improvement of ECppk1:
EPVM is a gene which can generate polyP in E.coli BL21.
We inserted ECppk1, vgb and mazE into the PBBR1MCS-2 vector to construct this part.
In bacteria, polyP is synthesized by polyphosphate kinase (PPK).
PPK1 can lengthen the polymer by using the γ-Pi phosphate bond of ATP,
and its reversible reaction is to synthesize ATP from ADP and Pi.
The ppk1 codes PPK1, which can promote the synthesis (major function) and decomposition (minor function)
of polyP with the residue of ATP. Therefore, we insert ppk1 gene into plasmid PBBR1MCS-2 in order to produce polyP.
Then we transformed the modified plasmid into E.coli BL21 and use synthetic wastewater to culture bacteria.
After 12 hours’ polymerization of phosphorus, bacteria are collected and freeze-dried for preservation.
We investigated the advantages of EPVM against normal E.Coli BL21 that only had PBBR1MCS-2,
especially the efficiency of synthesis. In other word, we want to use less bacteria to synthesize more polyP.
Therefore, we conducted the experiment above. Here are our results:
From the results above, it is not difficult to find that polyP productivity increased greatly,
thanks to the insertion of vgb and mazE genes.
Therefore, we realized the production of polyP in large scale and prepared raw materials for our future experiments.
2. The construction of vgb gene:
Bacterial hemoglobin (VtHb) is produced by the gram-negative bacterium, Vitreoscilla,
in large quantity in response to hypoxic environmental conditions.
The vgb gene coding for VtHb has been cloned in E. coli where it is expressed strongly by its natural promoter.
The expression of the vgb gene in Vitreoscilla is transcriptionally regulated by oxygen.
Vitreoscillu hemoglobin (VHb) is expressed at elevated levels in Vitreoscillu under oxygen-limited cultivation conditions.
The vgb can produce HGB so that we can increase the bacteria’s capacity of carrying oxygen,
which is beneficial to the growth of bacteria.
3. The construction of mazE gene:
Toxin-antitoxin (TA) modules are pairs of genes essential for bacterial regulation upon environmental stresses,
among which mazE is an important antitoxin.
The mazE protein is such a useful antitoxin that it can help bacteria grow longer and survive harsher environment,
thus producing more polyP. Thus, we insert mazE gene into E. coli genome to increase the productivity.
The mazE codes a kind of antitoxin protein, which can help the bacteria grow longer and produce more polyP.
4. The construction of vgb-mazE gene:
This part is the combination of vgb gene and mazE gene.
Adding vgb and mazE into the gene helps the growth of bacteria, thus improving the productivity of polyP.
PART3: APPLICATION PROSPECTS OF POLYP FROM LITERATURE READING AND INVESTIGATION
1. Prospects in medicine and health product
Pharmaceuticals companies can apply our ideas to develop a new drug curing IBD,
which can protect the intestinal epidermis under the inflammatory environment
thus making it difficult for the pathogenic bacteria to colonize. Owing to its distinctive mechanism,
it may also combine with current drugs to develop a cocktail therapy.
Manufacturers focusing on probiotic food can apply our ideas
to develop new probiotic food containing long-chain polyphosphate,
which may offer a sustainable protective effect on the intestinal epidermis and stabilize the balance of gut microbiome.
2. Prospects in environmental production
Environmental protection technology companies can apply our ideas to find a new solution for eutrophication,
which may establish a benevolent cycle of polyP application.
References
[1] Kyunghye Ahn and Arthur Kornberg, (1990), Polyphosphate Kinase from Escherichia coli, The Journal of Biological Chemistry.
[2] Zorzini, Buts et al., (2015), Escherichia coli antitoxin MazE as transcription factor: insights into MazE-DNA binding, Nucleic Acids Research.
[3] Kallio, P. T., Kim, D., Tsai, P. S., Bailey, J. E., (1994), Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions, The FEBS Journal.
[4] Kanak L. Dikshit, Rajendra P. Dikshit, Dale A. Webster, (1990), Study of Vitreoscilla globin(vgb) gene expression and promoter activity in E. Coli through transcriptional fusion , Nucleic Acids Research.