Team:Virginia/project-design

TRANSFOAM

Team Attributions
Description Design Experiments Model Safety
Results Future Directions Demonstrate
Parts Collection
Integrated Human Practices Public Engagement Collaborations Mid-Atlantic Meetup
Awards Medals

Introduction

Our Inspiration

When our team first came together, we went on a group hike at a nearby mountain range. During that trip, we learned we all shared a passion for our planet and were concerned for its future. Noticing our daily reliance on plastic, we came to see that our plastic-dependent lifestyles are not sustainable. This inspired us to learn more about how petroleum-based plastics are managed and how biodegradable plastics are produced. We wanted to resolve the disconnect between the usefulness of plastics and the harm that it wreaks on the environment.

PHB Production

Polyhydroxybutyrate (PHB) biosynthesis has been researched and optimized since the 1960s.1 Through the utilization of bacteria that possess the necessary biochemical pathways for a given carbon source (either endogenous or introduced) and grown in a nitrogen limiting medium, PHB accumulation can be easily achieved. Scale-up versions of PHB production, typically conducted in industrial scale bioreactors, have been studied and optimized since 1991.2 Bioreactors are optimized for production of bioplastics. Genetic engineering was introduced to PHB biosynthesis in the late 80s, as E. coli became popular for their simple genetic manipulation and well understood biochemical pathways. Metabolism of model carbon sources, such as glucose and other simple fats and sugars, soon became common products used for PHB synthesis, and this bioplastic production continued to bloom and advance towards becoming a household plastic.3

Biodegradable Plastic in Synthetic Biology

Methodologies developed to remediate polystyrene or styrene have been unsuccessful thus far, as many chemical modifications are required to convert it into a viable carbon source for metabolism in bacteria. There exists no organism which possesses both the biochemical pathway to breakdown styrene and to synthesize PHBs. Many organisms used for PHB production use only endogenous pathways to facilitate the biosynthesis of this plastic, and similarly most studies researching the biological degradation of styrene only utilize organisms with endogenous pathways. The lack of an organism without both pathways occurring naturally has prevented the use of a biological device to convert waste styrene to PHB. So, we decided to take biochemical pathways endogenous to bacteria optimized for both PHB production (Cupriavidus necator) and styrene degradation (Pseudomonas putida), and integrate them into E. coli to create a single biological device that can utilize styrene as a carbon source for PHB production. By using both pathways, needs for an environmentally friendly styrene remediation method and a less expensive carbon source for PHB production are solved using a single device.

Designing our Device

Choice of Chassis: Why we chose TG1

While E. coli K12 TG1 is not the most popular strain, it possess an intermediate pathway that is capable of converting phenyl acetic acid (the output of the sty gene) into acetyl-CoA (the input of the pha gene).4 The pathway endogenous to TG1 links the two plasmids together without having to insert another foreign plasmid which would further increase metabolic load on the bacterial cell.

In addition to being capable of completing the full, mapped-out pathway of styrene to PHB synthesis, the chassis also benefits from being a risk group 1 organism with minimal to no danger to the environment, or humans. The chassis is also capable of growing in the specified media (M9 minimal media) and being nitrogen-limited to elicit a PHB producing response. These factors fit perfectly into the puzzle being constructed, and made the TG1 strain an efficient chassis for this project.

Assembly Method

Golden Gate was used to assemble both plasmids. There were a maximum of 6 DNA parts that were synthesized for each plasmid, so it was important to use an assembly method that was effective for multiple parts. Golden Gate is efficient with this amount of parts (4-6) and thus was chosen, and successfully, used to assemble both plasmids. Our synthesized DNA inserts were designed for Golden Gate Assembly with the restriction site BsaI on each end, this ensures that all the pieces will assemble in the correct order.

How do the plasmids work together?

The sty plasmid works first to transport the styrene molecules into the cell, and then oxidize the styrene to phenylacetic acid through various series of enzyme-catalyzed reactions.5 Phenyl acetic acid is then taken through natural pathway of E. coli TG1 until reaching acetyl-CoA, where the pha plasmid converts it into PHB.6

Function

Maintaining a 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, we used a design similar to 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.7 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. Our plasmids also contained ampicillin (sty) and chloramphenicol (pha) to separately select either plasmid and could be used in a dual resistance antibiotic system as well if the kanamycin construct were to fail. 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 neo gene is split to produce proteins that are enzymatically active only as a dimer. One plasmid contains the N-terminal portion (neo-α), while the other plasmid contains the C-terminal portion (neo-β).7

Custom RBS

When introducing multiple genes into a nonnative species, these genes are likely not optimized transcription and translation in their new chassis. One common method to optimize genes is through codon optimization, a novel technique to improve protein expression level in living organisms by increasing translational efficiency of target gene. All of our coding sequence were optimized for E. coli. However, simply optimizing codons does nothing to increase the rate of translation in an organism. Our team decided to custom design the ribosomal binding site associated with each coding sequence on our BioBricks to increase the rate and efficiency of translation in our chassis. Denovo software uses software to analyze a coding sequence and optimize the translation initiation rate for each gene. Using DeNovo, we created synthetic RBS site for each gene and replaced the original RBS site with this new optimized one.8 Denovo uses software to analyze a coding sequence and optimize the translation initiation rate for each gene.

T7A1 promoters

The introduction of two plasmids bears a great metabolic cost to a cell. To decrease the energy wasted on translation in various parts of experimentation, we decided to use the T7A1 promoter to regulate expression of our two plasmids.This promoter is a tightly regulated, inducible, promoter that requires the presence of IPTG in the cell for translation to be initiated.9 Without the presence of IPTG, translation will not occur, but as soon as IPTG is added, translation ensues at a high rate. Coupling the T7A1 promoter with custom synthetic RBS sites allows for high levels of highly regulated expression, ensuring that production of introduced genes does not waste metabolic energy when they are not needed, but are overexpressed when transcription is induced.

“RNA polymerase subunits β and β′ are in two different shades of green, whereas the α N-terminal domains are in light and dark blue. The α-CTD, absent in the crystal structure, have been added as blue spheres. The σ subunit is in red. A simplified cartoon of the DNA is drawn to scale. The TS is in dark purple and the NTS is light purple. The contacts shown between the enzyme and the DNA reflect the decreased solvent accessibility of nucleotides in specific positions on the promoter during the formation of the complex as determined in this work by time-resolved hydroxyl radical footprinting.”9

Validation and Implementation

Plasmid inserts were synthesized in their entirety and cloned into backbones of respective antibiotic resistance. Once the plasmids were successfully assembled and transformed, restriction digests were performed and ran on gels to preliminarily confirm the identity of the plasmid. The plasmid’s identity was then accurately confirmed through sequencing.

References

  1. Sharma, M. & Dhingra, H. Poly-β-hydroxybutyrate: A Biodegradable Polyester, Biosynthesis and Biodegradation. British Microbiology Research Journal 14, 1–11 (2016)
  2. Blunt, W., Levin, D. & Cicek, N. Bioreactor Operating Strategies for Improved Polyhydroxyalkanoate (PHA) Productivity. Polymers 10, 1197–1197 (2018).
  3. Koller, M., Maršálek, L., de Sousa Dias, M. M. & Braunegg, G. Producing microbial polyhydroxyalkanoate (PHA) biopolyesters in a sustainable manner. New Biotechnol. 37, 24–38 (2017).https://www.ncbi.nlm.nih.gov/pubmed/27184617 (accessed Oct 20, 2019).
  4. Teufel, R. et al. Bacterial phenylalanine and phenylacetate catabolic pathway revealed. Proc. Natl. Acad. Sci. 107, 14390–14395 (2010).
  5. Lykidis, A. et al. The Complete Multipartite Genome Sequence of Cupriavidus necator JMP134, a Versatile Pollutant Degrader. PLOS ONE 5, e9729 (2010).
  6. Johnston, B. et al. The Microbial Production of Polyhydroxyalkanoates from Waste Polystyrene Fragments Attained Using Oxidative Degradation. Polymers 10, 957 (2018).
  7. Schmidt, C. M., Shis, D. L., Nguyen-Huu, T. D. & Bennett, M. R. Stable Maintenance of Multiple Plasmids in E. coli Using a Single Selective Marker. ACS Synth. Biol. 1, 445–450 (2012).
  8. Million-Weaver, S. & Camps, M. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked? Plasmid 0, 27–36 (2014).
  9. Salis, H. M., Mirsky, E. A. & Voigt, C. A. Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946–950 (2009).
  10. Sclavi, B. et al. Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter. Proceedings of the National Academy of Sciences 13, 4706–4711 (2005).
  11. San Millan, A. et al. Integrative analysis of fitness and metabolic effects of plasmids in Pseudomonas aeruginosa PAO1. ISME J. 12, 3014–3024 (2018).
  12. Using a codon optimization tool—how it works and advantages it provides. https://www.idtdna.com/pages/education/decoded/article/using-a-codon-optimization-tool-how-it-works-and-advantages-it-provides.