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From 2011.igem.org

Contents 1 Kevin 1.1 Characterisation of T7 - the S82-S75 system 2 Cyrille 2.1 Testing the pHM3 for the lost of the replication origin 2.2 Cloning into the old pHM3

Kevin

Characterisation of T7 - the S82-S75 system

To characterize T7 device, we decided to put on the same plasmid S82 and S75 in pSB1C3.
We'll do digestion XP / XS for S82 and S75 and ligation - transformation.

Cyrille

Testing the pHM3 for the lost of the replication origin

The sequencing showed that the pHM3 we have created has lost its replication origin for B. Subtilis along the cloning. To check the last step correct, all the backups of the pHM3 where minipreped again and digested in NcoI, EP and E.

NcoI is digesting inside the replication origin that has been lost, and in another place in the plasmid.

The results of the gel shows that we have lost the replication origin at the very begining fo the cloning, but we did not noticed it, because we have not tried it into Subtilis since.

The cloning is still possible in the old one but in XP only.

Cloning into the old pHM3

Cloning into the old pHM3 that we know that it is working. (S67)

The 6 clones of the vector was digested in XP and PCR purified, twice. (S67 DXP) . Standards yields.

The different inserts where digested in XP too, and gel extracted. I was cloning:

• pVeg KinA TT
• pVeg SpoVG RFP TT
• pVeg SpoVG ComS (H and B)
• pVeg T7 RFP TT
• pT7 T7 GFP
• TetO array

The cloning is ok, and the transformation worked, but there are lots of clones in the negative control and the manip needs an efficient cleaning.