Team:Edinburgh/Communication
Communication
The Joint Universal Modular Plasmid (JUMP) assembly : the novel vector platform for Golden Gate assembly.
Our constructs were assembled utilising a novel Golden Gate toolkit developed by Valenzuela-Ortega and French, known as Joint Universal Modular Plasmid (JUMP). Briefly, four base overhangs using PhytoBricks standard fusion sites were designed for a promoter part (P), ribosome binding site (R), N-terminal part (N), gene coding the protein of interest (O) and C-terminal tag (C) and a terminator sequence (T). Upon addition of the type IIS restriction enzymes, the individual parts can be assembled into level 0 vectors using BsmbI. These level 0 vectors were then subsequently assembled into level 1 expression vectors using BsaI.
The JUMP platform is a new potential tool for synthetic biology and may be included in the iGEM distribution kit in the future year. This video will help other teams in understanding JUMP and designing parts compatible to JUMP assembly
The modified Sequence Saturation Mutagenesis (SeSaM).
SeSaM is a relatively overlooked and undervalued random mutagenesis method which was formulated to build upon the mutational spectrum of epPCR. First developed in the Schwaneberg lab of RWTH Aachen University, its highly innovative design harnesses the use of 5’ biotinylated primers, the weakened phosphate backbone of α-thio deoxynucleotides, and the degeneracy of universal nucleotides. Essentially, the incorporation of α-thio deoxynucleotides such as dATPαS are randomly incorporated throughout the gene of interest in an initial PCR. After incubation with iodine, the PCR product is sheared at every position where there is a dATPαS. The 5’ forward biotinylated fragments are then isolated using streptavidin-coated magnetic beads from the other fragmented DNA and the reverse strand is melted away with a DNA melting solution. A universal base which is capable of base-pairing with any of the four canonical dNTPs is conjugated to the 3’ end of the forward fragment by terminal transferase. Two more subsequent PCRs generate the full length gene and replace the universal base with canonical dNTPs, ultimately generating a mutant library throughout the gene.
We develop an alternative method for a non-biased mutant library generation for enzyme directed evolution. This method provide more accessibility and more cost-effective to the potential SeSaM method.