Partnership

UTokyo partnership

Throughout our project we had extensive communication and collaborations with the iGEM team UTokyo, located in Tokyo, Japan. Their project is named Optopass. The following description was provided by UTokyo.

“Optopass is an Optogenetic Passcode System that can significantly contribute to the safe use and development of synthetic biology. Within the system, a sequence of different color inputs works as a cryptogram to express the genes of interest. We have designed a model in which the security is released by exposing S. cerevisiae to red, blue, and UV light in the correct order and thereby triggering the desired metabolic pathway.”

On this page you can read about our shared project objectives, our modeling collaboration, and our system design collaboration.

Shared objectives

In discussion with UTokyo, it was clear that we had mainly two shared objectives. The first was choosing optimal polymer lengths. In our project MOD³ we wanted to find protein linker lengths that would result in the highest possible protein-protein reaction rate. UTokyo found that they could use the programs we had constructed for examining their system consisting of DNA and recombinase. While examining and choosing polymer length was a part of both of our projects, we optimized protein linker lengths while different DNA lengths were examined in Optopass.

The second shared objective was regarding DNA binding methods. Our projects involved several different binding methods, and we agreed to share our results and insights to support each other. The common goal of using DNA binding mechanisms also meant that we could have meaningful discussions regarding project design.

Modeling collaboration

In our project, we wrote programs based on worm-like chain (WLC) modeling, which is used for modeling polymer bending. The designed programs were used to optimize protein linker lengths. You can read more about our modeling method and how the programs were used in our modeling section here.

The UTokyo team found another purpose for our programs. They modeled the contact probability of recombinases bound to DNA, and related their modeling results to the reaction rate of the recombinases. You can read more about how the WLC modeling was used in Optopass.

The way UTokyo used the modeling programs and their results is very informative as it shows how a program that is general enough can be a powerful tool in very different contexts. UTokyo’s modeling results were an inspiration for using our modeling approach to design a web app that is even more general and user-friendly. You can read more about the app here.

System design collaboration

We had an extensive discussion with UTokyo regarding the parts of our projects involving proteins binding to DNA. In MOD³, dCas9 and zinc-fingers were used in the cell-free and cell-based part respectively. In Optopass the following DNA-binding proteins were used: synTALE, EL222, and CcaR. Both our team and UTokyo researched different DNA binding methods when designing our projects.

Zinc-fingers and synTALE binding strength

UTokyo compared the binding strength of zinc-fingers and synTALE to DNA, which was beneficial to both projects. A high binding strength is very desirable in MOD³. In the cell-based part of the project, when zinc-fingers bind to DNA a transcription factor is released. However, two zinc-finger proteins must bind simultaneously to the same DNA strand. This means that it would be a problem if zinc-fingers do not bind strongly to the DNA, as the probability of simultaneous binding would be lower. You can read more about the cell-based project here.

UTokyo tested binding strength by using mCherry. mCherry is a red fluorescent protein whose expression can be measured using fluorescence spectroscopy. In the UTokyo project, the expression of mCherry was regulated by synTALE and zinc-fingers binding to DNA. The same target site was used for both proteins, consisting of 18 base pairs. The target site is located in the TDH3 promoter for mCherry. When either zinc-fingers or synTALE would be bound to the target site, expression of mCherry would be inhibited. UTokyo measured how mCherry expression was affected by both zinc-fingers and synTALE by comparing to the expression when neither zinc-fingers or synTALE was present, the control. The figure below shows that using synTALE resulted in less expression than zinc-fingers or the control. This indicates that synTALE has a higher binding strength to DNA. The results show that using synTALE instead of zinc-fingers in MOD³ could also result in higher binding strength, and therefore a better system.

Scaffold RNA

The option of using scaffold RNA (scRNA) was also a topic of discussion. UTokyo, who initially thought of using dCas9 and scRNA, suggested that scRNA could improve the design of the cell-free part of MOD³. In the cell-free part, dCas9 proteins were combined with two different RNA sequences, one for each binding site. If you have not already read about the cell-free part, we refer you to this page before continuing. dCas9 proteins are connected with RNA strands that determine binding sites for the dCas9. In our project there are two binding sites, with about 30 base pairs between them, and two corresponding RNA strands. For each RNA strand, the corresponding dCas9 protein was connected to a specific TEV half. As the two different RNA strands correspond to two different TEV halves, the TEV halves can combine when two dCas9 proteins bind to the same DNA strand. There is however a problem with the design, which occurs if dCas9 proteins switch RNA strands. Two dCas9 proteins that bind to the same DNA strand could then be connected to the same type of TEV half, either cTEV or nTEV, which would reduce the efficiency of our tests. If scaffold RNA were to be used instead, the RNA strands connected to dCas9 molecules would also be bound to other proteins, that in turn are connected by protein linkers to TEV halves. The proteins binding to the RNA could for example be MS2 coat proteins. Then dCas9 proteins exchanging RNA would not be an issue, as one RNA strand would always be connected to a specific TEV half. Using scaffold RNA was however not implemented in MOD³, but it remains as a possible improvement of the design.