Team:Uppsala/Description
Table of contents
Project overview What we are aiming for
We are developing a method to make CRISPR and microfluidics more available to iGEM teams and researchers. The technique will be used to fuse a fluorescent protein called UnaG to a genomic protein in both prokaryotes and eukaryotes. We are including state of the art research involving the CRISPR associated protein CPF1 and microfluidic methods.
UnaG is a fluorescent protein that needs bilirubin as a co-factor in order to fluoresce. Bilirubin binds non-covalently, which facilitates the possibility of creating inducible fluorescent switches from UnaG. Bilirubin occurs naturally in higher vertebrate cells, making it suitable as a biosensor for research on vertebrates. It is therefore convenient to use UnaG together with CRISPR systems, such as CRISPR/CPF1.
CPF1 cuts downstream of the PAM sites and leaves 5’ overhangs. By providing UnaG with the complementary overhangs, we could insert this fluorescent protein in the genome of our host. In order to increase the chances of correct insertion, we aim to engineer UnaG with homology arms, which enables cells to insert UnaG by means of homologous recombination in the exact position where the genome has been cut.
To facilitate the insertion of the genomic material we will design a microfluidic chip capable of transformation. This will be done through soft lithography by 3D printing a mold and baking a PDMS chip on it. A microfluidic chip will reduce the amount of reagents needed to perform a transformation, which could potentially reduce the cost and workload of a conventional transformation. The chip methods are not size-dependent, therefore it will be possible to do any given plasmid insertion with the same device.
By using this chip, cell transformation becomes simpler and cheaper to do for other iGEM teams and small laboratories. In our project we will use it along with CRISPR to fuse UnaG with a genomic protein in yeast, but a microfluidic chip could potentially be used for any transformation technique.
Project origins
The Uppsala team was started up in January with introductory meetings and brainstorming of project ideas. The iGEM Uppsala association recruited two project leaders from earlier iGEM Uppsala teams. These project leaders were then in charge of recruiting and leading the team itself. Anyone interested in iGEM were welcome to the introductory meeting and to help with the brainstorming. The potential projects were researched further to decide on the viability of each idea as an iGEM project. Researchers and university staff that have been involved in the iGEM Uppsala project earlier years were invited to give input on the ideas. With their feedback and questions in mind, further research was done.
Day of Decision
The 7th of April was decided as the day of Decision, when we would irrefutably chose a project . During the weeks before Decision day, projects with low interest were abandoned to allow more research on other potential ideas. As the day of Decision was upon us, only four projects remained. The team decided to choose project by majority decision, where the chosen project has to have more than 50% of the votes. Only team members voted in a closed poll, and as the project leaders held the selection process they decided to exclude themselves from the decision. After the first round, two of the project ideas differed by only one vote. The other two projects received one and no votes respectively. These two were removed from the next round of voting. The votes were read up one by one, which led to much excitement. The projects took turns of being in the lead, but when all votes had been read, one project was chosen with nine votes against seven. This project, CRISPR on a Chip, was what would fill our days and dreams the following months. Obviously the decision was celebrated by the team going out together for dinner at one of Uppsalas student pubs.
The months leading up to the summer
With only one project to focus on, the research picked up a new pace. Taking a lesson from what was learnt by last years team, the first round of gene synthesis was planned for the middle of May. This would hopefully allow us to have what we need when we received access to a lab. Having 18 students in the team required good organization and smaller working groups. With this in mind, three lab groups were put together by mixing biology, biotechnology and chemistry students in each group. Further research were done in these lab groups, though research came to a halt right before lab start due to exams. At the 7th of June we stepped into the course lab that we were assigned, as the 6th of June is a national holiday in Sweden. While making buffers and preparing competent cells, a complete plan for the summer was set up.
