Team:Bulgaria/IMPROVE
We improved the gRNA expression vector pSB1C3-gRNA - part BBa_K2515002 from the Registry. It was originally designed by the iGEM Bulgaria 2017 team for easy cloning and expression of gRNAs in E. coli. Nevertheless, it is a high copy number vector with no easy option for a plasmid curing. To overcome these limitations, we PCR amplified the pSC101-based thermosensitive origin of replication from the pE-FLP vector (Plasmid #45978). Next, we developed an aqua cloning-based approach to substitute the original pSB1C3 origin with this new part. The resulting vector was low copy number (that is sufficient for gRNA expression due to the high efficiencies of the CRISPR/Cas9 systems) and can be eliminated by cultivation at 42 o C.
Link: http://parts.igem.org/Part:BBa_K2515002
NB! The generated vector is a low copy number plasmid, thus, one needs to use reduced antibiotic concentrations and the colony growth requires more time. In addition, the pSC101 ori contains a SpeI restriction site and, therefore, is not BioBrick RCF10- compatible! If you need to use it – adopt the aqua cloning procedure from the part’s page in the Registry.
We submitted a new part BBa_K2847002 – it contains the thermosensitive replication origin and can be used as a template in PCR amplification reactions.
An illustration of our concept for a part improvement. The normal origin of replication found in pSB1C3 was substituted by the pSC101* T-sensitive origin. The resulting vector was a low copy number plasmid and required decreased antibiotic levels for growth (left). If needed, it could be successfully cured by an overnight cultivation at 42 o C (right).