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Cloning Strategy
Introduction Freiburg standard
Cloning with the conventional iGEM Biobrick Standard (RFC 10) does not allow the cloning of fusion proteins because a stop codon is inserted between the fused parts. Thus the Freiburg standard (RFC 25) was established by Kristian Müller, Katja Arndt and Raik Gruenberg in 2007 by adding the AgeI and NgoMIV restriction sites. This enables cloning of in-frame fusion proteins. expression vector
pEx Vector
As we needed a suitable expression vector, we altered the NEB vector pMAL-p5x according to our needs. Via PCR the chloramphenicol acetyle transferase (CAT) was amplified out of a lab vector. Add-on-tail primers were used for the PCR containing the prefix XbaI and NgoMIV and the suffix AgeI, SpeI, NotI and PstI. EcoRI and NotI were excluded so that the ribosome binding site (RBS) stays within a distance of 7 nucleotides from the start codon. The add-on-tail primers also included the RBS GAAA ressembling the highly efficient shine dalgarno sequence of Biobrick part: BBa_B0030. In front of the prefix MfeI and behind the suffix HindIII were inserted to enable the exchange of the MalE gene with the CAT gene. [[image:Freiburg09 Cloningstr1.jpg|Fig1. pEX Vector|300x300px]] pJS418 and pJS419 Vectors
To perform a co-transformation it is neccessary to use vectors with different antibiotica resistence genes. The two pJS vectors were suitable because they both carry the CAT gene which provides the bacteria with a chloramphenicole resistence whereas the expression vector pEx includes a ampicilin resistence gene. Another reason why we choose the pJS vectors are the differently strong Shine-Dalgarno sequences. The Fok_a constructs were hard to express in E. coli so we needed a vector with a weak RBS. pJS419 is the vector who fits to that, so we was able to transform and proliferate Fok_A in this vector. pJS419 is inducible by IPTG, like pEX, another reason to pick them for the co-transformation. pBAD Vector
The pBAD vector enables the cloning of parts that already contain a ribosome binding site as the vector itself has none. The vector carries a ampicilin resistance gene. Cloning strategy
The basic parts were ordered from Mr.Gene or used from last years Freiburg iGEM team (also from Mr.Gene) and were delivered in the vector pMA (Link????) which is fully compatible to RFC 10 and RFC 25. The vector carries a ampicilin resistence gene and an E. coli ori. To avoid ligations with small parts like Tags or linkers, we adapted our cloning strategy. Thus we always cut out the larger inserts with NgoMIV and PstI, whereas vectors with smaller inserts were cut open with AgeI and PstI. As first step we joined tags and binding proteins in one pMA vector and linker and Fok constructs in another pMA respectively. In the second step the tag-binding_protein was cloned into the expression vector pEx followed in the third step by linker-Fok construct. Triple ligations were used to accelerate the cloning process. Therefore the vector was cut open with XbaI and NgoMIV, insert one was cut with XbaI and AgeI and the second insert with NgoMIV and AgeI. The strategy requires a ....
