Team:Virginia/Experiments
To see the protocols we used during experimentation, click here.
To see the team’s lab log, click here.
DNA Synthesis
Design using Teselagen
Using Teselagen, a DNA design and plasmid assembly software, our team designed out BioBricks for Golden Gate and Gibson assembly respectively.
pha Plasmid
Our plasmid that encodes genes for the biosynthesis of polyhydroxybutyrate was designed with 3 essential genes, phaA, phaB, and phaC. A customized synthetic RBS site was inserted before each coding region, to optimize translation. The genes phaA (BBa_K3192015), phaB (BBa_K3192016), and phaC(BBa_K3192017) encode for enzymes essential for the conversion of acetyl coa into polyhydroxybutyrate (PHB). phaA encodes for b-Ketoacyl-CoA thiolase. phaB encodes for b-Ketoacyl-CoA reductase. phaC encodes for poly(3-hydroxybutyrate) polymerase. This plasmid was maintained with chloramphenicol resistance.
paa Pathway
We chose a chassis organism, E. coli TG1, that constitutively expresses the paa pathway. Implementing the paa pathway through a plasmid into a chassis organism would be too large because there are 11 genes essential to the conversion of phenylacetic acid to acetyl-coA. We chose TG1 so that we did not have to incorporate this pathway into our chassis.
sty plasmid
Our second plasmid encodes for the biodegradation of styrene into phenylacetic acid. 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. This plasmid was maintained with ampicillin resistance. Using two different antibiotic resistance genes, one for each plasmid, allowed for both to be maintained within a single cell.
Assymbly & Plasmid Confirmation
Golden Gate
Golden gate assembly was used to assemble both the pha and the sty plasmids. All of our synthesized DNA constructs were designed to come together in the exact order that was desired, through the addition of a 5 base pair recognition sequence. This sequence is complementary on each end with the adjacent overlapping sequence.
Retrieved from 1.
The PHA plasmid was cloned with fragments that were synthesized by Twist and IDT to have the desired restriction sites on either end of the parts. These parts were then assembled into the vector pGGA provided by NEB. This has chloramphenicol resistance and was grown on LB - Agar plates. The sty plasmid was also synthesized by Twist and IDT to have the correct restriction sites on either side of the fragments. This was assembled with the vector (pGGEV_3_Linker) that contained the ampicillin resistance gene. Both plasmids have different origins of replication to ensure that they can both be maintained during replication.
Gibson Assembly
Gibson Assembly was attempted for both parts. Due to the amount of parts and the nature of Gibson assembly, it was difficult to assemble the plasmid in the correct order and prevent reannealing in one reaction tube. This is the trademark for Gibson Assembly, thus we found that it was not a viable solution for ensuring correct plasmid formation. Many attempts of various methods were tried to prevent reannealing during the Gibson Assembly reaction, but for the amount of parts in the tube, it was difficult to ensure all the parts formed in the correct order, and all the pieces were contained within the plasmid.
Pictures taken from GenScript GenBuilder Plus Cloning Kit, that our team used to attempt Gibson assembly.
Restriction Enzyme Digest and Gel Electrophoresis
To confirm the identity of our assembled plasmids, the plasmid was digested with 1 to 2 enzymes that would produce unique bands when run on a gel. The digested plasmid was run on a gel to confirm its identity to see if the bands matched up with what was expected from the digest. Teselagen was again used to analyze the expected result of the digest and our resulting digest.
Teselegan Gel Electrophoresis simulation results of pha plasmid cut with restriction enzyme EagI
Gel Electrophoresis results of pha plasmid cut with restriction enzyme Eagl
Sequencing the Plasmids
Once a gel was run and indicated that our plasmid likely assembled correctly, the plasmid was sent out for sequencing. Primers for each plasmid were designed to ensure that high quality results would be obtained from the overlaps in assembly to ensure that one piece assembled adjacent to its next piece. The primer for each overlap began sequencing ~150 base pairs upstream from the overlap.
Electroporation for Chassis Assembly
Electrocompetent TG1 cells were transformed with the assembled pha plasmid using electroporation. In order to insert the second sty plasmid into the TG1 cell containing the pha plasmid, the TG1 cells had to be made electrocompetent. Antibiotic selection was used to ensure the first pha plasmid was still retained within the chassis. Once we confirmed that the cells were again electrocompetent, and the pha plasmid was retained, the bacteria were transformed with the assembled sty plasmid using electroporation. These cells were then selected with ampicillin and chloramphenicol dual resistance agar plates.
Our two plasmids in the TG1 chassis growing on a dual resistance chloramphenicol and ampicillin plate.
PHB Production & Extraction
Integrating Styrene
Styrene cannot be added directly to the medium the cells are growing in, so an alternative method to introducing styrene needed to be used. Consulting the literature, there were various methods to introduce styrene to our cells. Two promising methods were vaporization1 and partitioning.2 Partitioning was chosen due to the ability to measure the amount of styrene uptake with gas chromatography.
Styrene was dissolved in dioctyl phthalate, an organic solvent, which was then placed above the M9 medium in a biphasic mixture.
The method of partitioning is done by the solute, styrene, distributing through two immiscible solvents, dioctyl phthalate and M9 medium. When shaken, the styrene molecules will partition into the water molecules of the M9 medium. Because styrene is very insoluble to water, only a small, but sufficient, amount will reach the cell membranes. There is an equilibrium of styrene concentration in both solvents, and when styrene molecules are taken into the cell, this change in concentration will continue to push styrene towards equilibrium as it is metabolized. This will provide the cells with a consistent influx of styrene. These 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 phenyl acetic acid.
Red Nile Staining
Red Nile is a qualitative means to determine PHB production within a culture of cells.3 Red Nile is effective in measuring lipid droplets by fluorescence microscopy and flow cytofluorometry. The fluorescence is conditional on the presence of strong hydrophobic regions, hence why it is ideal for detecting PHBs. Our team utilized Red Nile staining and fluorescence microscopy to preliminarily assay PHB production within our chassis to confirm the function of our PHB plasmid.
SDS Extraction
Sodium Dodecyl Sulfate (SDS) is a common chemical used for cell digestion and washing. PHB producing cells were spun down and SDS extraction was performed on the pellet to separate the cell pellet from the PHB. SDS digests the cell such that PHB can be separated from It is important to note that the SDS extraction does not physically separate the cell debris and PHB, only lyses the cell.4
Chloroform with Acetone
We first used hot acetone (60°C) to lyse the cells open, exposing the PHB contained within the cell. Chloroform was then added to this mixture. Chloroform is an efficient solvent for PHB, and can be used to purify it from a solution. Dissolution of the pellet in chloroform followed by filtration allowed PHB to be separated from cell debris. PHB could then be reprecipitated into solution with the addition of methanol. Repetition of this procedure multiple times was found to increase purity of PHB extraction.5
SDS and Chloroform in Conjunction
Using a sequential extraction of SDS and chloroform was implemented by our team to attempt to lyse the cells with SDS extraction, and then purify the PHBs from solution with chloroform extraction. The conjunctive procedure allowed efficient separation of cell debris and PHB as well as increased PHB purity from extraction.
Sonication and centrifugal differentiation
Given the environmental focus of our team, SDS, but especially chloroform extraction were not appealing to us, as both involve potent and harmful chemicals. Our team searched for another protocol for PHB extraction with lower environmental impacts, and eventually developed our own using the density difference of PHB and organic cell debris to separate the two. Through sonication, lysing the cells using sound energy, we broke the cells apart to release PHB. Applying the pellet obtained from sonication to a sucrose solution with a density of 1.23 g/mL the PHBs could be separated from the cell pellet through density differential centrifugation. The sucrose density was chosen because the density of PHBs is 1.25 g/mL and the density of the average cell debris is 1.10 g/mL, so by centrifuging the pellet in a solution with a density slightly less dense than PHB the two solids can be effectively separated.
This protocol is outlined in more depth in our protocols handbook.
This protocol has undergone refinement, but is still far from perfection. We recognize that there were likely impurities in our PHB samples of cell debris dense enough to pellet with it, and PHB that was likely light enough not to pellet with the rest of the pellet.
References
- Ward, P. G., Goff, M., Donner, M., Kaminsky, W. & O’Connor, K. E. A Two Step Chemo-biotechnological Conversion of Polystyrene to a Biodegradable Thermoplastic. Environ. Sci. Technol. 40, 2433–2437 (2006).
- Osswald, P., Baveye, P. & Block, J. C. Bacterial influence on partitioning rate during the biodegradation of styrene in a biphasic aqueous-organic system. Biodegradation 7, 297–302 (1996).
- Tyo, K. E., Zhou, H. & Stephanopoulos, G. N. High-Throughput Screen for Poly-3-Hydroxybutyrate in Escherichia coli and Synechocystis sp. Strain PCC6803. Appl. Environ. Microbiol. 72, 3412–3417 (2006).
- Chen, Q. & Zhang, L. H. Study on Synthesis of PHB by Moderate Halophile and Aqueous Extraction of PHB. Applied Mechanics and Materials (2014) doi:10.4028/www.scientific.net/AMM.448-453.160.
- Hahn, S. K., Chang, Y. K. & Lee, S. Y. Recovery and characterization of poly(3-hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombinant Escherichia coli. Appl. Environ. Microbiol. 61, 34–39 (1995).
