Part:BBa_K1702002
E. coli fadD and fadL under control of lac promoter
This part is a composite of the fadD (BBa_K1702000) and fadL (BBa_K1802001) parts.
Details about fadD:
Acyl-CoA synthetase is the first enzyme the beta oxidation pathway. It attaches Coenzyme A to long chain fatty acids, allowing them to continue through the pathway. The transcription of fadD is controlled by the enzyme produced by fadR under standard conditions.
Details about fadL:
LCFAs are naturally moved into E. coli by a long-chain fatty acid outer membrane porin, which is encoded for by fadL.
Detail about P_lac+fadD+fadL construct
By placing both fadD and fadL under the control of an inducible promoter, we can control expression. The combination of fadD and fadL should greatly increase E. coli's uptake of fatty acids into the cell.
Experimental Characterization
The intended use of this part was to increase beta-oxidation, which is the natural process for the breakdown of fatty acids, in order to allow for frying oil waste to be used as an alternative substrate. We ran a couple experiments on this part. For exact experimental protocol see our wiki. The first experiment was a growth on 0.04% frying oil suspended in M9 with the assistance of a detergent (Brij 58). This experiment got slightly odd data and can not be used to definitively say what was occurring.
The second experiment we ran was on more concentrated frying oil to create a functional prototype. When grown on 50% frying oil we were able to measure the growth using optical density. As a general rule the fadD+fadL construct did better than the control (the lac promoter). However there are some fluctuations in the data that don't really make sense.
When grown in 100% frying oil the cell growth measurement was a little more complicated. Initially we tried to use OD but the spectrometer could not handle the media. So we switched to cell weight. This method of measurement is very noisy. The viscosity of the oil makes it very hard to work with. There is lots of room for error, including: getting a consistent volume of oil in the draw, spinning down the sample all the way to collect all the cells, and pulling off all the oil so only the cell weight is left. Our results indicate at least an initial observation that the addition of these constructs is beneficial to cell growth.
The data that actually says more about the cells’ usage of the oil is the qualitative data. As the experiment proceeded the color of the samples changed. From this we conclude that the cells were doing something to alter the composition of the media. Additionally when no cells were added the water separated from the Brij 58:oil solution into very clear layers. A little mixing quickly re-homogenizes the sample. After about two hours of growth the media separated significantly less and by the 12 hour data point stayed homogenous even without shaking. This told us that the media was becoming more soluble in water, implying that the cells took up and used the insoluble fatty acids.
As a less noisy measurement was counting colony forming units in dilutions. This is a much more exact measurement.
We believe that this construct performed worse than the fadL by itself due to the added metabolic stress of producing two enzymes instead of just one.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2939
- 1000COMPATIBLE WITH RFC[1000]
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