Team:WashU StLouis/Design

Design

Designing a Super Cell

Our team set out to design an E. coli strain that overproduced ATP and electron donors in order to create a suitable environment for nitrogenase activity, as well as increasing the activity of other cellular functions requiring ATP or reduced electron donors. In order to accomplish this goal, we partitioned the project into three phases:

  1. Project Design: Identifying genes of interest (GOI), constructing plasmids, and conducting further modeling (Flux Balance Analysis)
  2. Experiments: Transforming the plasmids into DH10B (wild-type E. coli) and measuring for our gene products
  3. Proof of concept: Demonstrate the functionality of the plasmids

Project Design

We began by exploring the literature and academic papers on this topic. From our literature reading, we identified three genes that we believed would help us overproduce intracellular ATP and two genes to overproduce reduced electron donors.

ATP Genes

The three ATP-producing genes were pck, pgk, and the ATP synthase multi-gene operon

  • pck: Codes for phosphoenolpyruvate carboxykinase (PCK), a kinase involved in gluconeogenesis that decarboxylates oxaloacetate (OAA) into phosphoenolpyruvate (PEP) and creates ATP in the process
  • pgk: Codes for Phosphoglycerate kinase (PGK), a kinase involved in glycolysis that converts 1,3-Biphosphoglycerate to 3-phosphoglycerate by removing a phosphate group and creates ATP in the process
  • The ATP synthase operon (genes atpA - atpH): These genes code for their respective subunits of ATP synthase, a multi-protein complex that works to produce ATP when on a proton gradient. Unfortunately, ATP synthase was later dropped due to size constraints in cloning.

All genes were extracted from E. coli MG1655.

We believed that overexpressing genes that transcribe ATP-producing enzymes would lead to a cell with more intracellular ATP.

Electron Donor Genes

The two electron donor genes were fldA and petF

  • fldA: Codes for flavodoxin, an electron-transfer protein native to E. coli
  • petF: Codes for ferredoxin, an electron-transfer protein native to Synechocystis

E. coli’s natural electron donor is flavodoxin. Organisms that have nitrogenase activity, diazotrophs, use ferredoxin, a related electron donor, to reduce nitrogenase in order to fix nitrogen gas. We wanted to see if ferredoxin could be produced in E. coli and how it would perform relative to the naturally occurring flavodoxin at various levels of expression.

We extracted fldA from E. coli MG1655 and petF from Synechocystis 6803.

We believed that overexpression of these genes would lead to more reduced electron-transfer proteins available for various cellular uses.

In addition to fldA and petF, we also identified pfo as a metabolically relevant. It encodes for pyruvate flavodoxin/ferredoxin oxidoreductase (PFO). PFO is the actual enzyme that reduces flavodoxin/ferredoxin in their respective systems. Check out our modeling page to learn why we decided to use this gene and the process of modeling of our Super Cells.

Below are the basic plasmid maps:

Each group of plasmids had a different inducible promoter so we could fine tune the expression of our GOIs to make sure the cell was not overburdened to the point of preventing the desired behavior.

  • pBad: a promoter that is induced by the presence of arabinose (Ara) was used for the ATP plasmids
  • pTet: a promoter that is induced by the presence of anhydrotetracycline (aTc) was used for the electron donor plasmids
    • Additionally, for each of the electron donor genes (fldA and petF), we created a version of the plasmid with and without pfo (4 plasmids total)

We used golden gate assembly to construct the plasmids, with BsaI as the Type IIS restriction enzyme. We designed all primers using SnapGene and ordered them from IDT. Check out our protocols page to learn more

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.
  2. Emmerling, M. et al. Metabolic flux responses to pyruvate kinase knockout inEscherichia coli. J Bacteriol 184, 152–164 (2002).
  3. von Meyenburg K., Jørgsen B. B., van Deurs B. (1984). Physiological and morphological effects of overproduction of membrane-bound ATP synthase in Escherichia coli K-12. EMBO J. 3 1791–1797.
  4. BLASCHKOWSKI, H. P., KNAPPE, J., LUDWIG-FESTL, M. and NEUER, G. (1982), Routes of Flavodoxin and Ferredoxin Reduction in Escherichia coli. European Journal of Biochemistry, 123: 563–569. doi:10.1111/j.1432-1033.1982.tb06569.x

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