Part:BBa_K3192025
genes styABCDE
Degradation of Styrene
This BioBrick takes five essential genes from Pseudomonas putida, a bacteria capable of naturally metabolizing styrene to degrade it to create a device capable of insertion into another bacterium to give any chassis the ability to metabolize styrene. Bacteria expressing this BioBrick can use styrene as a carbon source for metabolism. These genes have been codon optimized for expression in E. coli K12 chassis. The 2019 Virginia iGEM’s addition to the T7 registry (BBa_K3192012) was used as the inducible promoter that was regulated by the presence of IPTG. IPTG added to the growth media induced high levels of transcription and translation to express the coding sequence of styABCDE (For more information about the promoter see its part page).
The genes styA, styB, styC, styD, and styE are essential for proteins and enzymes that uptake styrene into a cell and degrade it to phenylacetate. styA (BBa_K3192015) Codes for the styrene monooxygenase subunit A, which catalyzes the conversion of styrene to styrene oxide. styB (BBa_K3192016) codes for styrene monooxygenase subunit B, which catalyzes the conversion of styrene to styrene oxide. styC (BBa_K3192017) codes for epoxystyrene isomerase which catalyzes the reaction from styrene oxide to phenylacetaldehyde. styD (BBa_K3192018) codes for phenylacetaldehyde dehydrogenase which catalyzes the reaction from phenylacetaldehyde to phenylacetic acid. styE (BBa_K3192019) codes for the putative styrene transporter which is a membrane bound protein that aids in the transport of styrene across the cellular membrane and into the cell. The biochemical pathway associated with all these enzymes can be visualized below converting styrene into phenylacetate.
Integrating Styrene into Growth Media
Styrene cannot be added directly to the medium the cells are growing in, so an alternative method to introducing styrene needed to be used. This plasmid was expressed in E. coli TG1 cells, and grown in M9 minimal media, styrene and dioctyl phthalate. The method of partitioning is done by trapping molecules of styrene into the insoluble water molecules of the M9 medium. Styrene was dissolved in Dioctyl Phthalate, an organic solvent, which was then placed above the M9 medium in a biphasic mixture. When shaken, the styrene molecules will move out of the organic solvent and get trapped within the water molecule structures.These trapped styrene molecules can then be transported into the cell using the membrane-bound transporter protein translated by the styE gene. Once taken into the cells, they can begin breaking down the styrene into phenylacetate.
Measuring Growth in Styrene
After this part had been assembled, we transformed it into E. coli DH5-ɑ. The DH5-ɑ were cultured in M9 growth media supplemented with 2% styrene and 400µL of glucose. 400 µL of glucose was chosen for addition because it is not enough to solely sustain bacterial growth.1 Growth was measured through OD taken periodically. The graph below displays the OD of cell growth over 48 hours. The increase in OD to 0.349 supports that the culturing cells are able to survive with styrene as, essentially, their sole carbon source, indicating that our plasmid is functional.
References:
1) Ward, P. G., Roo, G. de & O’Connor, K. E. Accumulation of Polyhydroxyalkanoate from Styrene and Phenylacetic Acid by Pseudomonas putida CA-3. Appl. Environ. Microbiol. 71, 2046–2052 (2005).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2440
Illegal NheI site found at 5360
Illegal NheI site found at 5383 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1062
Illegal BamHI site found at 3841
Illegal BamHI site found at 5017
Illegal XhoI site found at 1 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 143
Illegal NgoMIV site found at 3001
Illegal NgoMIV site found at 3368
Illegal AgeI site found at 3191 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 3779
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