Difference between revisions of "Part:BBa K3192029"

Latest revision as of 00:07, 22 October 2019

sty plasmid with neokanamycin resistance

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.

Depiction of the styABCD pathway for the degradation of styrene to phenylacetate.

Use of Alpha Neo-kanamycin to dual plasmid system

Keeping two plasmids in a culture that is continually growing can sometimes be difficult. Ensuring that both plasmids are doubled and split equally on fission is important. To do this, the 2019 Virginia team used a split antibiotic system developed by iGEM 2017 Vilnius’s plasmid control design. Having both plasmids contain a gene to produce a partial protein for kanamycin resistance, and combining together to form a full resistance complex was the goal of the plasmid design. This would regulate resistance towards the bacteria that weren’t capable of producing both plasmids, and ensured that the resistance would continually select for those that maintained both plasmids during fission. The kanamycin construct would hypothetically be more efficient for long-term culturing of bacteria, in terms of stifling the effects of evolution for a longer time than would a dual resistance system.

The BioBrick BBa_K2259018 developed by Vilnius in 2017 contained resistance for half of the kanamycin antibiotic. Virginia iGEM used this BioBrick to maintain this part in the same chassis as BBa_K3192031, which possessed the BioBrick BBa_K2259019, conferring the second half of the kanamycin resistance.

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.

Styrene integration through a biphasic mixture of styrene dissolved in dioctyl phthalate and cells grown in M9 Minimal Medium.

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.

This graph depicts the measurement of optical density over time for a culture of E. coli TG1 transformed with this assembled gene cluster.

Sequence and Features