Difference between revisions of "Part:BBa K1807002"

Latest revision as of 03:06, 22 October 2021

Escherichia coli Polyphosphate Kinase Enzyme

This part is a protein generator device producing the Polyphosphate Kinase (PPK) gene from Escherichia coli.

Usage and Biology

PPK produces polyphosphate, more specifically PPK catalyzes the reversible conversion of the γ-phosphate of ATP to the end of the polyphosphate chain (Akiyama et al., 1992). This enzyme is responsible for the formation of long chain polyphosphate molecules (up to a thousand orthophosphate residues long) - see illustration below.

The E. coli PPK coding sequence was derived from the pBC29 plasmid, published by Akiyama et al., 1992. iGEM York 2015 obtained pBC29 with Dr Jay Keasling's kind assistance.

Akiyama, Masahiro, E. Crooke, and Arthur Kornberg. "The polyphosphate kinase gene of Escherichia coli. Isolation and sequence of the ppk gene and membrane location of the protein." Journal of Biological Chemistry 267.31 (1992): 22556-22561.

Sequence and Features

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, so the data can provide some reference to this part.

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.

iGEM2021_Qdai Experiment

We constructed a plasmid (pBEST-ppk) containing BBa_K1807002 and measured the growth rate of bacteria by O.D.₆₀₀ using medium with normal phosphorus concentration and medium with double phosphorus concentration.
After the O.D.₆₀₀ readings reached a plateau, the increase in phosphorus uptake by pBEST-ppk was characterized by measuring the decrease in the amount of phosphorus in the medium using the QuantiChrom Phosphate Assay Kit (BioAssay Systems). The results showed that pBEST-ppk showed high phosphorus uptake both in normal medium and in medium with twice the phosphorus concentration.

Fig.1 Phosphorus uptakes (mg) in LB medium.

Fig.2 Phosphorus uptakes (mg) in medium with twice the phosphorus concentration.