Team:Virginia/Entrepreneurship

Manifold

MANIFOLD

Index:

Entrepreneurship

Kickstarting Virginia iGEM’s First Entrepreneurship Committee

Starting an entrepreneurship committee was a natural calling for the Virginia iGEM 2020 team – with the launch of iGEM’s new EPIC committee and increasing concerns regarding IP protection, the establishment of an entrepreneurship committee was a no brainer. There was a rising necessity for a space where our teammates could hold discussions and debates addressing our countless questions on commercialization, patentability, and more importantly, the implications of our product. Essentially, our team needed a space where we could hone our skills in creative problem solving, negotiation, and networking in the business sector. We needed to develop a culture which carefully balanced the open-source innovative nature of iGEM. With enough research, informational interviews, and inspiration from iGEM EPIC, we were able to justify the creation. At the time of a global pandemic and national public health crisis, we craved to be even more intentional about our work than before, and entrepreneurship offered the perfect opportunity to ignite this passion.

The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of

bacterial microcompartments

Bacterial microcompartments (BMCs) are organelle-like structures, consisting of a protein shell that encloses enzymes and other proteins. BMCs are typically about 40–200 nanometers in diameter and are entirely made of proteins. The shell functions like a membrane, as it is selectively permeable.

(BMCs) with encapsulated dsDNA scaffolds

[1]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis. The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of bacterial microcompartments (BMCs) with encapsulated dsDNA scaffolds that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis.

Our Journey with IP Protection

Questions about what constitutes a public disclosure first arose in conversation between the wet lab and human practices committees. Bridging the gap between our team, the university, and the Charlottesville (Cville) community required openly sharing our thoughts and ideas to seek expertise and collaboration on MANIFOLD. However, we had to be aware of the implications that public disclosures would pose if we had any plans on commercializing our foundational advance in the future. Our lack of expertise in the space in combination with our curiosity to learn more led to our reaching out to lawyers at Cville BioHub—Cville’s diverse biotech center.

Nikki Hastings, the Executive Director of Cville BioHub, was fundamental in helping the team understand how to become better connected with the Charlottesville community while simultaneously protecting our technology, while lawyers David Vance and Rahul Keshap helped us to navigate startup law.

Vance helped us to understand the ins and outs patent filing logistics. He shared his expertise about biotech startups and gave us some key insight into the startup landscape in Cville and iGEM. Insights gathered included:

A public disclosure constitutes any non-confidential disclosure of our idea or invention in a public environment. This meant one-on-one Zoom calls with experts and mentors would not be an issue, but it would be smart on our part to file for a Provisional Patent Application (PPA) a few weeks before the iGEM wiki freeze
Can we write the PPA ourselves? Absolutely! We should draw thorough diagrams illustrating our invention and learn from previous biotech parents to map out our PPA. We should also refer to the official Manual of Patent Examining Procedure (MPEP) to ensure our work is lawful

Vance enabled us to take charge of our own IP protection and we could not have done so without his help.

Keshap, on the other hand, was important in helping us to understand corporate law. He gave us great advice on how to run a team like a business, and consider the future impact of the decisions we made. Insights included:

It is important to clearly define leadership roles within the team-- team members wear multiple hats in a startup, but the ultimate responsibility must belong to someone to get the work done
Vested equity is a technique where equity is ‘vested’ or secured over time-- this way, teammates will have to earn their equity over time by adding evident value to the team

Fig 1. Figure taken from iGEM Tainan 2019 for demo purposes. Notice how the figure is much longer than it is wide, and two images are coupled together to achive this. Try to do that as well so it looks good.

Additionally, protein linkers are usually present between this nucleic acid binding domain and the enzyme structure to prevent inhibition of enzyme activity. However the exact linker(s) used, if any, is(are) also dependent on the specific use case of the invention. These pathway enzymes are attached to the nucleic acid scaffolds via their nucleic acid binding domains. The nucleic acid recognition sequences (22) are unique or semi-unique sequences of nucleic acid monomers on the nucleic acid scaffolds to which the utilized nucleic acid binding domains have some degree of molecular complementarity. These nucleic recognition sequences comprise most of the scaffold and mark the locations to which the DNA binding domains of the pathway enzymes attach to the scaffolds. The nucleic acid spacers (32) are relatively short sequences of nucleic acid monomers that are also present on the nucleic acid scaffolds, between the recognition sequences. The linkage between the nucleic acid scaffolds (18) and protein shell (10) provides a means by which the nucleic acid scaffolds are bound to the protein shell through direct or multi-molecule complementarity. This linkage is found between the nucleic acid scaffolds and the protein shell. One example is through the addition of a nucleic acid binding domain (24) to one or more of the shell proteins forming a nucleic acid binding domain, shell protein fusion (14). Like the pathway enzymes, this nucleic acid binding-domain can be either internal to the shell protein structure or at its N or C terminus, where the exact placement depends on the shell protein being utilized. Alternatively, one or more intermediate proteins can be used to adhere the nucleic acid scaffolds to the shell, where the region of the protein interacting with the shell binds the shell via protein-protein complementarity (28) with a given shell protein, and the region of the protein interacting with the nucleic acid scaffold binds another recognition sequence on the nucleic acid scaffold through another nucleic acid binding domain (30). This forms a shell protein binding, nucleic acid domain fusion (26).