Generator

Part:BBa_K3022003

Designed by: Zhanyu Gao   Group: iGEM19_Nanjing-China   (2019-09-12)


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.

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