Part:BBa_K2135000

pck

TL;DR: pck codes for Phosphoenolpyruvate carboxykinase (Pck), a kinase involved in gluconeogenesis in E. coli. Specifically, PCK decarboxylates oxaloacetate (OAA) into phosphoenolpyruvate (PEP) under gluconeogenic conditions.

Work with overexpression of pck has been shown to inhibit cell growth [Na et al, 2014].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 97
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 250
  Illegal AgeI site found at 817
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 613
  Illegal SapI.rc site found at 166
  

Usage and Biology

pck codes for Phosphoenolpyruvate carboxykinase (PCK), a kinase involved in gluconeogenesis in E. coli. Specifically, PCK decarboxylates oxaloacetate (OAA) into phosphoenolpyruvate (PEP) under gluconeogenic conditions. This reaction results in the creation of ATP, which the cell can now use for various pathways throughout the cell. The reaction is displayed below:

Other uses: Work with overexpression of pck has been shown to inhibit cell growth [1]. Cell growth was inhibited because normal cell metabolism was interrupted by the overproduction of reactive oxygen species, like peroxide or superoxide. The growth inhibited cells though do have an excess amount of intracellular ATP, which can be harnessed for a desired cellular function. For example, the [http://2016.igem.org/Team:WashU_StLouis WashU_STL 2016 team] used pck to create a cell that overproduces ATP with the hopes of incorporating nitrogenase, an ATP consuming enzyme, into the cell and boosting the enzyme’s activity. Additionally, this part can be used to create a cell that overproduces ATP in order to produce more recombinant proteins.

Below is a graph indicating the luminescence produced by a cell with a construct containing pck and an inducible promoter (Arabinose). As seen, the highest induction level resulted in the most ATP production. While it is not statistically significant, it does show that pck can effect the intracellular concentration of ATP. DH10B is a wild-type control.

References

1. Na, Yoon-Ah, Joo-Young Lee, Weon-Jeong Bang, Hyo Jung Lee, Su-In Choi, Soon-Kyeong Kwon, Kwang-Hwan Jung, Jihyun F. Kim, and Pil Kim. "Growth Retardation of Escherichia Coli by Artificial Increase of Intracellular ATP." Journal of Industrial Microbiology & Biotechnology 42.6 (2015): 915-24.