Team:HokkaidoU Japan/Test

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Dr. E.coli : The smallest protein injector in the world

There are several transfection methods to introduce nucleic acid into cells, however methods to introduce proteins directly into cells are complicated.

Unlike introducing DNA, which changes cell genetic makeup permenantly, proteins can act at pinpoint acuracy. And afterwards be degraded leaving no permanent information in the target cell.

Our mission was to find an easy way to introduce desired proteins in the cell. Speficaly into eucaryotic cell. In order to do this we chose Type 3 Secreation System. Find in Salmonella and EPEc. These bacteria are pathogenic and working with them is problematic. To make things easier and safer we ordered DNA fragment which contains T3SS`s syringe part. And introduce it into E.coli.

We also needed protein to secreat. We desined it by ataching secreation signal to desired protein. Our choice was GFP. Also added NLS to make it go to nucleus.

Currently we will test tjis system on column cancer cells to see if GFP will localize into nucleus.

HokkaidoU ToolBox

This is the first year that HokkaidoU team is joining iGEM. It was a journey full of errors and new experieneces. And most of the protocols we used we were unfamiliar with.

At the begining we decided to amplify most of the parts by PCR and not miniprep. This introduced it's own chalenges. So we would like to share some of the tools we thought useful in the procces.

Restriction Enzyme Digestion Visualization primers

When you are cutting out insertion from the vector you can see 2 bands in electrophoresis and see instantly if digestion was succesful. Not so with the parts PCRed with prefix and suffix primers. These are only possible truly universal primers. But we wanted to see if restriction was a success. To distinguish cut offs they must be big enought. So we ventured into vector to find some universal primer sites.Things get complicated because there are more than 40 vectors in registry. We couldn't tailor one set of primers for all but we did it for curent asembly standart and few other vectors.

This primers could also be used for checking how various primers effect restriction enzymes. OR for purification small parts like RBS.

Easy 3 piece ligation program

This is program which calculates the neccesary reagants for restriction and ligation. You only have to choose well address, vector, PRC primers, concentration after PCR and part to plasmid ratio.