These numbers have to mean something, right?
After assembling and sequence confirming all six plasmid constructs (pck, pgk, fldA, fldA-pfo, petF, and petF-pfo), we transformed them into DH10B E. coli. Additionally, we transformed all four electron donor plasmids into ΔaceE E coli. We then experimented with these cell cultures to find out if our gene products were being produced. Detailed procedures for each experiment can be found on the Experiments page.
In order to determine the amount of intracellular ATP, we used a luminescence assay. Firefly luciferase is an enzyme that produces luminescence and requires ATP. The amount of intracellular ATP initially produced could be determined by adding luciferase and luciferin (the substrate) to our ATP producing cells and measuring the amount of luminescence produced. As seen in the graph below, pck seemed the best candidate for the construct that produced the most ATP.
Additionally, we sent our pck and pgk plasmid constructs to Cardiff University's iGEM team to validate our results. Similarly to our initial measurements, pck produced more ATP than pgk and was the most promising ATP-producing construct.
Electron donor determination:
In order to determine the amount of electron donors, the absorbance of the cell extract was measured from 200nm to 700nm. Both ferredoxin and flavodoxin have characteristic peaks in this region. Specifically, ferredoxin shows peaks around 330nm and 430nm, with reduced ferredoxin showing a small peak at 545nm in the absence of the 430nm peak. Flavodoxin shows peaks around 350nm and 450nm. The exact placement of these peaks varies throughout the literature 1,2,3. As seen in the graphs below, our electron donor producing cells seem to show the characteristic peaks of their respective electron donor. Specificaly, in the graph on the right, there is a small hump near 430nm for the petF cells at the highest inducer level, indicating that ferredoxin was present in these cells. Additionally, in the graph on the left, which shows our ΔaceE cells, there is a peak in the 300-350nm region, indicating that flavodoxin was present (not ferredoxin because E. coli do not naturally produce ferredoxin). This graph also shows that our modeling prediction, that ΔaceE cells should have increased electron donors due to the knocking out of a competing pathway, was correct. Not all graphs are shown.
In addition, 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 does not naturally produce and therefore regulate. The color may come from a large amount of cytochrome d, another electron transport protein which has a red color when it is reduced4. The tube on the left is petF cells at the highest induction level and the tube on the right is a DH10B control.
In order to see if the produced electron donors could be utilized by the cell, a proof of concept experiment was conducted.
- Jenkins, Christopher M., and Michael R. Waterman. "Flavodoxin and NADPH-flavodoxin reductase from Escherichia coli support bovine cytochrome P450c17 hydroxylase activities." Journal of Biological Chemistry 269.44 (1994): 27401-27408.
- Bottin, Hervé, and Bernard Lagoutte. "Ferrodoxin and flavodoxin from the cyanobacterium Synechocystis sp PCC 6803." Biochimica et Biophysica Acta (BBA)-Bioenergetics 1101.1 (1992): 48-56.
- Medina, Milagros, et al. "A laser flash absorption spectroscopy study of Anabaena sp. PCC 7119 flavodoxin photoreduction by photosystem I particles from spinach." FEBS letters 313.3 (1992): 239-242.
- 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.