Team:USP-EEL-Brazil/Proof
Now is time to give you the answers for the questions we asked you and ourselves, actually.
Does tocopherol indeed help with cell growth?
To ask this question, we had the cell growth analyzed with oleic acid in the medium and a same medium with oleic acid and tocopherol. We then measured cell density in the plate reader. Once again, one problem would be the higher concentrations of oil would distort our readings.
This problem was adressed by making the 2:1 solution of oleic acid:ethanol. This solution did not fully stop the separation of the phases, but greatly reduced it, and with the sample in the shaker, it would be well shaken and well mixtured, meaning the nutrients and oil should be available to the cells. The nutrients would no longer stick to the tube wall. The downfall of this method is the presence of ethanol in the medium, which at a maximum of 5% v/v concentration shows no harm at all for the bacteria, but higher concentrations may be toxic
The same ammount of ethanol was also added to the control samples, to simulate the ethanol toxicity and compare only the growth relative to oil and tocopherol.
All the graphics' proportions from now on represent tocopherol|oleic acid added in uL.
Examining the graphic, we note that the sample with 1:1 tocopherol:oleic acid concentration, the 50|, is the one which had the highest growth rate, meaning that our theory of tocopherol helping the bacterial cells to whitstand the oil concentrations may prove itself right. We can also clearly see that the excess of tocopherol helped to inhibit our 2|1 sample. It is noteworthy that the 0.032M sample had a better growth rate than the control, showing already some potential even in the 1:2 proportion.
This graphic shows exactly the same behaviour as the previous one. We can clearly see the 1:1 curve standing tall above the others, followed by the 1:2 and then the control. The other two samples are inhibited by the excess of oleic acid. We believe that the weird point in the 10h mark of the control curve might be result of experimental error, even tough we can't pinpoint exactly where the error might have occurred.
So now, to our last sample wave, we have the 0.128M of oleic acid ones. We had a little problem with sample handling and unfortunately lost a 1|8 sample in the process. Instead of re-doing the whole experiment, we decided it wouldn't have much value, since it would certainly have its curve somewhere near the control and 1|40 curves. We also did not make any 1|1 samples, since this quantity of substrate would exceed our 5% limit for ethanol concentration in these samples. Besides these absences we can once again see the 1|2 sample towering the other curves and we can also suppose that a 1|1 would have this behaviour too.
These results give us total confidence to affirm that tocopherol present in a rich fatty medium have a pretty relevant role in protecting the cell and stimulate cell growth very effectively proving our initial idea of synthethizing tocopherol to protect the cells.
Sources
Okai, Y.; et.al. Protective effects of alpha-tocopherol and beta-carotene on para-nonylphenol-induced inhibition of cell growth, cellular respiration and glucose-induced proton extrusion of bacteria (2000).
ALBERMANN, C. et al. Biosynthesis of the vitamin E compound delta-tocotrienol in recombinant Escherichia coli cells. ChemBioChem, v.9, n.15, p.2524-2533, 2008.
Washington iGEM. Make it or break it. Avaible at:https://2011.igem.org/Team:Washington
Hino, T.; Andoh, N.; Ohgi, H. Effects of beta-carotene and alpha-tocopherol on Rumen bacteria in the utilization of long-chain fatty acids and cellulose (1993)
