Difference between revisions of "Team:Uppsala"

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.