Part:BBa_K3731004
EPVM-PBBR1MCS-2
EPVM-PBBR1MCS-2 is a plasmid which can generate polyP in E.coli BL21. We inserted ppk1, vgb and mazE into the PBBR1MCS-2 vector to construct this part.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 5166
Illegal suffix found in sequence at 1 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 5166
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 5172 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 5166
- 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 5166
Illegal suffix found in sequence at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 5166
Illegal XbaI site found at 5181
Illegal SpeI site found at 2
Illegal PstI site found at 16 - 1000COMPATIBLE WITH RFC[1000]
iGEM2021_Nanjing-China Experiment
Group: Nanjing-China 2021
Author: Hao Yin
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 transduce the modified plasmid into E.coli BL21 and use synthetic wastewater to culture bacteria. After 12 hours’ polymerization of phosphorus, E.coli are collected and freeze-dried for preservation.
we investigated the advantages of EPVM against normal E.Coli BL21 that only has PBBR1MCS-2, especially the efficiency of synthesis. In other word, we want to use the less bacteria to synthesize more polyP and the polyP’s molecular weight is more accurate. Therefore, we conducted the above experiment. 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.
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