Difference between revisions of "Part:BBa K3731004"
 
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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.</p>
 
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.</p>
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<p>Therefore, we conducted the above experiment for the first time. Owing to the lack of experience, our experiment time span is too short to reflect the overall situation of polyP production efficiency. Here are our results:
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<p>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:
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[[File:T--Nanjing-China--Polyphosphate_OD600.png|800px|thumb|center|Figure1 values of OD600 against time in the polyphosphorous culture medium]]  
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[[File:T--Nanjing-China--OD600PA.png|800px|thumb|center|Figure1 values of OD600 against time in the polyphosphorous culture medium]]  
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[[File:T--Nanjing-China--LB_OD600.png|800px|thumb|center|Figure2 values of OD600 against time in the LB culture medium]]  
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[[File:OD600LB.png|800px|thumb|center|Figure2 values of OD600 against time in the LB culture medium]]  
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[[File:T--Nanjing-China--Polyphosphate_OD700.png|800px|thumb|center|Figure3 values of OD700 against time in the polyphosphorous culture medium]]
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[[File:T--Nanjing-China--OD700.png|800px|thumb|center|Figure3 values of OD700 against time in the polyphosphorous culture medium]]
 
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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. </p>
 
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. </p>
 
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Latest revision as of 07:07, 1 October 2021


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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 5166
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 5166
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 5166
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE 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
  • 1000
    COMPATIBLE 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:

Figure1 values of OD600 against time in the polyphosphorous culture medium
Figure2 values of OD600 against time in the LB culture medium
Figure3 values of OD700 against time in the polyphosphorous culture medium

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.