Difference between revisions of "Part:BBa K3022003"

 
 
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<partinfo>BBa_K3022003 short</partinfo>
 
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The  polyphosphate kinase in Citrobacter freundii ATCC 8090 is responsible for its intracelluar inorganic polyphosphate (polyP) production via reversibly catalyzing the transfer of terminal phosphate from ATP to a growing polyP chain. This organism is our chasiss, in which its native PPK1 will be overexpressed with a plasmid of medium-copy numbers.
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This part will be sub cloned into pSB1C3, a plasmid of high copy number, to yield pSB1C3-lac-CFppk1. Polyphosphate synthesis capability of C. freundii transformed with this plasmid will be subjected to compare with C. freundii that harbored medium-copy expressing vector pBBR1MCS2-CFppk1. In this way, we can demonstrate that why medium copy number is a better option of vector choice.
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Although PPKs from E. coli and C. freundii shares 96% identity, the C. freundii PPK1 has a glutamate and a lysine residue in positions 327 and 328, where E. coli PPK1 has much less strongly charged alanine and glutamine residues. These natural mutations of C. freundii PPK1 are distant from the PPK1 active site and found in interfaces between monomers of the PPK1 tetramer.This leads to a dramaticl increase of intracellular polyP accumulation.
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In addition of CFppk1, this part also includes the promoter and terminator sequences of the lac expression system, thus composing a CFppk1 expression cassette. As such, when it was sub cloned into a plasmid of certain copy number, we can get a relative precise result regarding plasmid copy-number’s effects on ppk1 expressing level and polyphosphate yields, rather than a promiscuous conclusion drawn from the differences of promoting and/or terminating strength imposed by differed plasmid expressing system.
  
 
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<partinfo>BBa_K3022003 parameters</partinfo>
 
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<h2>iGEM2019_Nanjing China Experiment</h2>
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<p>This year our team develops a simple solo medium-copy plasmid-based polyphosphate kinase (PPK1) overexpression strategy for achieving maximum intracellular polyphosphate accumulation.</p>
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<p>To test whether the C. f reundii derivative that wasconstructed on the basis of the solo medium-copy strategy could perform well in uptaking of exogenous Pi from the SMW, we compare DH5a, DH5a-MDPP and CF-MCPP</p>
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<p>
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Ps:
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SMW means Synthetic municipal wastewater</p>
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<p>DH5a-MDPP means dual-plasmid which contain high and medium copy DH5a ppk in DH5a</p>
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<p>CF-MCPP means solo medium-copy C. f reundii ATCC8090 ppk in C. f reundii ATCC8090</p>
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<p>DH5a-HCPP means solo high-copy DH5a ppk in DH5a</p>
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<p>CF-MDPP means dual-plasmid which contain high and medium copy C. f reundii ATCC8090 ppk in C. f reundii ATCC8090</p>
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<p>[[File:T--Nanjing-China--gold3.png|800px|thumb|center|Figure 1）supernatant Pi concentration in SMW]] </p>
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<p>[[File:T--Nanjing-China--gold4.png|800px|thumb|center|Figure 2）Comparison of each strain’s ability to removing P and COD consumption]] </p>
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<p>Reference:
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Wang X , Wang X , Hui K , et al. Highly Effective Polyphosphate Synthesis, Phosphate Removal and Concentration Using Engineered Environmental Bacteria Based on a Simple Solo Medium-copy Plasmid Strategy[J]. Environmental Science & Technology, 2017:acs.est.7b04532.</p>
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<p>Kato, J.; Yamada, K.; Muramatsu, A.; Ohtake, H. Genetic improvement of Escherichia coli for enhanced biological removal of phosphate from wastewater. Appl. Environ. Microbiol. 1993, 59 (11),3744−3749.</p>
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<p>Jones, K. L.; Kim, S.-W.; Keasling, J. Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria. Metab. Eng. 2000, 2 (4), 328−338.</p>
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<p>Liang, M. Z.; Frank, S.; Lunsdorf, H.; Warren, M. J.; Prentice,M. B. Bacterial microcompartment-directed polyphosphate kinase promotes stable polyphosphate accumulation in E. coli. Biotechnol. J.2017, 12 (3),1600415.</p>

Latest revision as of 15:07, 20 October 2019


lac-CFppk1 Cassette

This part will be sub cloned into pSB1C3, a plasmid of high copy number, to yield pSB1C3-lac-CFppk1. Polyphosphate synthesis capability of C. freundii transformed with this plasmid will be subjected to compare with C. freundii that harbored medium-copy expressing vector pBBR1MCS2-CFppk1. In this way, we can demonstrate that why medium copy number is a better option of vector choice.

In addition of CFppk1, this part also includes the promoter and terminator sequences of the lac expression system, thus composing a CFppk1 expression cassette. As such, when it was sub cloned into a plasmid of certain copy number, we can get a relative precise result regarding plasmid copy-number’s effects on ppk1 expressing level and polyphosphate yields, rather than a promiscuous conclusion drawn from the differences of promoting and/or terminating strength imposed by differed plasmid expressing system.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


iGEM2019_Nanjing China Experiment

This year our team develops a simple solo medium-copy plasmid-based polyphosphate kinase (PPK1) overexpression strategy for achieving maximum intracellular polyphosphate accumulation.

To test whether the C. f reundii derivative that wasconstructed on the basis of the solo medium-copy strategy could perform well in uptaking of exogenous Pi from the SMW, we compare DH5a, DH5a-MDPP and CF-MCPP

Ps: SMW means Synthetic municipal wastewater

DH5a-MDPP means dual-plasmid which contain high and medium copy DH5a ppk in DH5a

CF-MCPP means solo medium-copy C. f reundii ATCC8090 ppk in C. f reundii ATCC8090

DH5a-HCPP means solo high-copy DH5a ppk in DH5a

CF-MDPP means dual-plasmid which contain high and medium copy C. f reundii ATCC8090 ppk in C. f reundii ATCC8090

Figure 1）supernatant Pi concentration in SMW

Figure 2）Comparison of each strain’s ability to removing P and COD consumption

Reference: Wang X , Wang X , Hui K , et al. Highly Effective Polyphosphate Synthesis, Phosphate Removal and Concentration Using Engineered Environmental Bacteria Based on a Simple Solo Medium-copy Plasmid Strategy[J]. Environmental Science & Technology, 2017:acs.est.7b04532.

Kato, J.; Yamada, K.; Muramatsu, A.; Ohtake, H. Genetic improvement of Escherichia coli for enhanced biological removal of phosphate from wastewater. Appl. Environ. Microbiol. 1993, 59 (11),3744−3749.

Jones, K. L.; Kim, S.-W.; Keasling, J. Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria. Metab. Eng. 2000, 2 (4), 328−338.

Liang, M. Z.; Frank, S.; Lunsdorf, H.; Warren, M. J.; Prentice,M. B. Bacterial microcompartment-directed polyphosphate kinase promotes stable polyphosphate accumulation in E. coli. Biotechnol. J.2017, 12 (3),1600415.