CONTRIBUTION

Overview

Throughout our exploration of PHB production and secretion system, our team was able to make a number of contributions to the field of bioplastic production as well as the growing realm of synthetic biology. The following lists our most significant contributions from this our project ESPHA: an extented collectivized literature review regarding PHA bioplastics as a whole and PHA synthesis mechanisms, a model for PHB accumulation for production scaling, new parts for generalized and PHB-specific secretion models, and an early characterization of vesicle nucleating peptides (VNps).

PHA

Extended Literature Review

Throughout the progression of our project, we often found ourselves immersing in various literature reviews and analyzing numerous data from past iGEM teams and field-specific research. Due to the large complexity of PHA production, we have collectivized all of our references and resources into a single extended literature review. We hope future teams as well as research groups may find our organized information helpful for their projects and explorations.

Our Results

In Project ESPHA, we have demonstrated the applied integration of PHB production with effective secretion methods. We have analyzed and compared results from a number of secretion systems, including the type I Hlya secretion system, type II TorA secretion system, and VNps. Our gathered results may be used in future models of PHB producing systems in E. Coli. Further, our research explores the potential for reducing downstream production costs of PHB and other types of PHA production models via the modification of the extraction stage, paving a path towards a future of PHA production that is less costly, environmentally friendly, and scalable.

Model for Scaling

One of the key focuses of project ESPHA has been scalability. While our research delved into the specific mechanisms of PHB secretion, our larger context for our project is to demonstrate the potential for PHB and other bioplastics to compete in the plastic market. In doing so one of the most important aspects of PHB production is the cost to yield ratio for largescale models. Through Human Practices, we have reached out to several stakeholders in the bioplastic and plastic industries for their input and perspectives into the current field. However, in addition to that, we have developed a model for comparing PHA accumulation with respect to the change in carbon and nitrogen levels. In other words, our model examines how fast carbon or nitrogen levels in cell cultures diminishes according to differing PHA accumulation rates. Therefore, the model can be used to as a framework for largescale PHA production involving carbon and nitrogen-based resources for industrial microbial cultures. Our constructed model can be found on the Modeling page.

Secretion Systems

Parts

Adding on to the growing collection of BioBrick parts in the registry, we have contributed a total of 3 new basic parts and 21 new composite parts this year. While we have utilized most of the secretion tags for secreting PHB for the purposes of our project, these secretion tags and systems could be modified to accommodate a variety of other uses including, but not limited to, alternative proteins, and drug delivery systems. Additional documentation of our used and designed parts can be found on the Parts page.

Our Results

In Project ESPHA, we have demonstrated the applied integration of PHB production with effective secretion methods. We have analyzed and compared results from a number of secretion systems, including the type I Hlya secretion system, type II TorA secretion system, and VNps. Our gathered results may be used in future models of PHB producing systems in E. Coli. Further, our research explores the potential for reducing downstream production costs of PHB and other types of PHA production models via the modification of the extraction stage, paving a path towards a future of PHA production that is less costly, environmentally friendly, and scalable.

Vesicle Nucleating Peptides (VNps)

One of our most promising contributions this year was our use and documentation of vesicle nucleating peptides (VNps). Our team discovered the potential use of VNps after coming across an article published by Eastwood et. al describing a novel system in which proteins–such as those that are typically challenging to produce in E. Coli–could be exported through membrane-bound vesicles with a single peptide tag called VNps. Not only are VNps a viable alternative, they provide further advantages to traditional secretion systems as well. As previously implied, VNps allow for the isolated production and storage of proteins with insoluble, toxic properties. Vesicles also provide a stable, long-term environment for proteins. VNps have been proven to possess numerous flexible characteristics. Eastwood et. al, originally explored the possibility of improving the tag itself by making simple modifications to the VNp amino acid sequence. Their results found that they could enhance protein yields, enhance vesicular export over a wider range of culture temperatures, and shorten the sequence to just 20 residues while working in a variety of E. Coli strains. Further, VNps are compatible with a variety of plasmids, promoters, and induction levels. Moreover, the vesicle system can be used for the expression of a wide range of recombinant proteins.

Through our project, we have successfully characterized many of these features. Firstly, we have confirmed the functionality of VNps in our BL21 DE3 E. Coli strain. In addition, we have demonstrated the implementation of VNps in a practical application of PHB secretion via a VNp-Phasin-GFP fusion protein design. While we utilized the VNp6 variant in particular for our PHB secretion model, we have provided parts designs for two other promising variants, VNp2 and VNp15. We have added all three VNp basic parts designs to the registry while acknowledging the fact that the parts were uploaded to the Parts Registry by ASIJ-Tokyo iGEM but have been designed by an outside lab. We hope future teams and research may apply similar if not innovative methods for utilizing vesicle nucleating peptides to their designs.