Team:HokkaidoU Japan/Test

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Latest revision as of 07:18, 24 October 2010

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 accuracy and afterwards be degraded leaving no permanent information in the target cell.

We tried 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. Found 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. As BAC vector came already inside a E.coli we were spared the enormous task of purifying and transforming it to the E.coli by ourselves.

We also designed a protein to secrete. For secretion the chimeric protein has to have secretion signal used in T3SS, we chose SrlP. To visualize the process and to make sure it did secrete we added GFP as a reporter. Arabinos promoter to switch it on on demand. And to make it even more interesting, a nuclear localization signal.

We tested this on rabbit colon cancer cells.

We think this technology will applicable by introducing proteins to the nucleus to fight cancer or induce normal cell to be stem cell.

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 using standard assembly protocol after restriction enzyme digestion to acquire insert of the plasmid you can clearly see two bands in the electrophoresed gel. Not so with the parts PCRed with prefix and suffix primers. They very effective because they are the only 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 got 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. And 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. It contributes greatly to the speed and precision of biobrick assembly.

@@ Line 10: / Line 10: @@
 <br>
-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,
+Unlike introducing DNA, which changes cell genetic makeup permenantly, proteins can act at pinpoint accuracy and afterwards be degraded leaving no permanent information in the target cell.
 <br>
-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 tried 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. Found 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. As BAC vector came already inside a E.coli we were spared the enormous task of purifying and transforming it to the E.coli by ourselves.
 <br>
-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.
+We also designed a protein to secrete. For secretion the chimeric protein has to have secretion signal used in T3SS, we chose SrlP. To visualize the process and to make sure it did secrete we added GFP as a reporter. Arabinos promoter to switch it on on demand. And to make it even more interesting, a nuclear localization signal.
 <br>
-Currently we will test tjis system on column cancer cells to see if GFP will localize into nucleus.
+We tested this on rabbit colon cancer cells.
+<br>
+We think this technology will applicable by introducing proteins to the nucleus to fight cancer or induce normal cell to be stem cell.
+<br>
 == 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.
 <br>
-Restriction Enzyme Digestion Visualization primers
+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.
-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.
 <br>
- This primers could also be used for checking how various primers effect restriction enzymes. OR for purification small parts like RBS.
+=== Restriction Enzyme Digestion Visualization primers ===
+When using standard assembly protocol after restriction enzyme digestion to acquire insert of the plasmid you can clearly see two bands in the electrophoresed gel. Not so with the parts PCRed with prefix and suffix primers. They very effective because they are the only 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 got 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.
+<br>
+This primers could also be used for checking how various primers effect restriction enzymes. And for purification small parts like RBS.
+<br>
 === 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.
+  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. It contributes greatly to the speed and precision of biobrick assembly.