Team:WashU StLouis/Proof

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Proof of Concept

Measuring biotin production to calculate reduced electron donors

Since the results of the electron donor absorbance data showed promising evidence of electron donor production, we decided to go a step further to ensure that our electron donor constructs were producing reduced and usable electron donors. We performed an assay which showed that not only were the products being overproduced, but also that there was significant data supporting the claim that these electron donors were usable in E. coli's metabolism.

Biotin, commonly know as vitamin B7, is produced via a pathway that uses reduced flavodoxin/ferredoxin,specifically the SAM pathway that turns dethiobiotin to biotin1. Specifically, there is a 1:1 stoichiometric relationship between reduced electron donors and biotin molecules produced2. If we measure the amount of biotin in our cells, we will indirectly measure the amount of reduced electron donors present in our cells. We used a readily available biotin assay to conduct our proof of concept experiment.

Results showed that overall, our constructs worked to increase biotin production, as seen in the graphs below.

  • When our constructs were in DH10B cells, the fldA, fldA-pfo, petF, and petF-pfo strains produced more biotin than the control DH10B strain at some induction level
  • When our constructs were in the ΔaceE knockout cells, the fldA, fldA-pfo, and petF strains all produced more biotin than the control DH10B strain, control ΔaceE knockout strain, and DH10B+constructs strain (petF-pfo data is not present because it was not able to grow)

Additionally, we saw a phenotypic change in our electron donor cells. Post-induction, the cells that contained the petF or petF-pfo constructs had a distinct red color. The red color is likely due to the increased amounts of ferredoxin in the cell, which E. coli is obviously not use to. The color may come from a large amount of cytochrome d, another electron transport protein which has a red color when it is reduced3.

This experiment illustrates a proof of concept for our Super Cells. By showing that our cells could overproduce biotin, we have shown that the theory behind the Super Cells work: that overproducing certain co-factors will increase the amount of resulting product

Go to the Conclusions page for a complete summary of our findings.

References

  1. Lin, Steven, and John E. Cronan. "Closing in on complete pathways of biotin biosynthesis." Molecular BioSystems 7.6 (2011): 1811-1821.
  2. Picciocchi, Antoine, Roland Douce, and Claude Alban. "Biochemical characterization of the Arabidopsis biotin synthase reaction. The importance of mitochondria in biotin synthesis." Plant physiology 127.3 (2001): 1224-1233.
  3. Haddock, B. A., J. ALLAN Downie, and P. B. Garland. "Kinetic characterization of the membrane-bound cytochromes of Escherichia coli grown under a variety of conditions by using a stopped-flow dual-wavelength spectrophotometer." Biochemical Journal 154.2 (1976): 285-294.