From 2007.igem.org

Contents 1 Mon, 23rd July to Sat, 04th August 2007 2 Thu, 21st June to Friday 20th July 2007 3 Wed, 13th June to Wed, 20th June 2007 4 Thu, 19th April to Thu, 24th May 2007

Mon, 23rd July to Sat, 04th August 2007

We made a PCR to get Calmoduline coding regions with 2, 4 and 6 Glycins linker on each side of the calmoduline region to clone it between the splitted lactamase fragments
the 320d blac1-wz-blac2 was cutted with KpnI and SpeI and after several test digests ligated with calmoduline (including the different glycine linkers)
this final construct was run through another background test in ampicilline which, again, was completely positive even without adding any calcium to the solution
the rest of the work is listed beneath labnotes 3

Thu, 21st June to Friday 20th July 2007

Transformation of the 320d blac1-wz-blac2 Plasmid into RV-308 E.coli cells didn´t show any growth so we made another transformation with XL-1 blue E.coli cells which was positive
a test digest with HindIII and SpeI showed good results and we sequenced the Plasmid (positive!) and made a glycerine stock
we also made a background test on some Ampicilline plates, which was positive and showed growth on every plate. so we knew that the lactamase was active even the parts were seperated!

Wed, 13th June to Wed, 20th June 2007

Amplification of the positive sequenced 320d blac1-wz-blac2(without periplasmic sequence) for glycerine stocks
due to we wanted to test our system in vivo, our coach reminded us that we would need a periplasmic signaling sequence in front of our splitted enzyme. so we made a new try to ligate a 320dp blac1-wz-blac2 plasmid

Thu, 19th April to Thu, 24th May 2007

Ligation of the 300d / 300dp blac 2 insert into the 310d / 310dp blac 1 vectors via NheI and HindIII, to create the 320d / 320dp blac1-wz-blac2 plasmids
only the plasmid without signaling sequence was ligated, the other one didn´t show good results in the test digests, so we put it aside
Amplification of the 320d blac1-wz-blac2 (without periplasmic signaling sequence)
to test the plasmid we made another digest with HindIII and SpeI, which was positive so we sequenced it and got a nearly perfect accordance

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