Team: iGEM Frankfurt -


BioBrick Assembly

The BioBrick plasmid pSB1C3 was sent as a linear fragment with blunt ends. The problem is that we wanted to transform this plasmid into E.coli to get a higher amount of it. Therefore we made a blunt end ligation to transform the circular plasmid into E.coli. After plasmid preparation we get a higher amount of the plasmid but when we wanted to digest pSB1C3 to assemble the BioBrick parts only a small amount of it had been linearized.

So after a few trys we decided to take the BioBrick device with the red fluorescent protein (RFP). We transformed it into E.coli and made a plasmid preparation. Then we digest it as well and put it together with our digested BioBrick fragments. After ligation we transformed it again into E.coli. After some colonies were grown we didn't see the expected differences of colonies that take the RFP insert again and colonies that contain our wished BioBrick device. So we put the agar plates into the fridge. After some our we looked for it again and we could see very well the differences between the red and the white colonies.

The control digestion of our expected BioBrick devices shows that we get BioBricks of the following parts:

1. Protein

  • HMG-CoA-Reductase
  • FPP-Synthase
  • bifuntional Cyclase
  • ent-Kaurene Oxidase

2. Promoter

  • pHXT7
  • pPGK1
  • pPFK1

3. Terminator

  • tHXT7
  • tPFK2
  • tCYC1

After we have made a "Midi-Preparation" we also give the BioBricks away for sequencing reaction. The results here show, that the terminator and promoter BioBricks have no insert. So we now have to check if these BioBrick parts really have no insert.
All four proteins show positive results.
However we leave all BioBrick parts in the parts registry with the suitable sequences until we have checked our results again.