Part:BBa_K1945001:Design
Homologous Recombination Plasmid for Synechocystis sp. PCC 6803
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 734
Illegal suffix found in sequence at 764 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 734
Illegal SpeI site found at 765
Illegal PstI site found at 779
Illegal NotI site found at 740
Illegal NotI site found at 772 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 734
- 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 734
Illegal suffix found in sequence at 765 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 734
Illegal XbaI site found at 749
Illegal SpeI site found at 765
Illegal PstI site found at 779
Illegal NgoMIV site found at 1269 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1384
Design Notes
We wanted to create a new iGEM plasmid derived from pSB1C3 to make working in Synechocystis sp. PCC 6803 more convenient. There are a handful of iGEM teams working with Synechocystis sp. PCC 6803, and the existence of this plasmid would allow other teams working with this organism to have a homologous recombination plasmid ready to clone any sequences into following iGEM assembly standards. Very few plasmids replicate within the organism, so HR is the preferred method.
We've chosen the neutral site slr1068 as it doesn't have any illegal restriction sites.
Between VF2 and VR, we've inserted neutral site sequences with BioBrick prefix and suffix cloning sequences in between so that the any sequence between the neutral sites will be integrated in the genome of Synechocystis sp. PCC 6803 through HR. The plasmid is still essentially pSB1C3, just the prefix and suffix have been moved within the neutral site sequences. Previous teams have attempted to create HR plasmids, but their neutral sites had illegal restriction sites; they were not released for us to use. Also, to remain compliant with Registry standards, other teams placed the prefix and suffix outside the neutral site. This means that other methods such as Gibson cloning would be needed to insert desired sequences.
The design of our pSB1C3 HR plasmid preserves the integrity of iGEM BioBrick assembly. Using just EcoRI, XbaI, SpeI, and PstI restriction enzymes, any team should be able to clone successfully rather than need to resort to other methods for assembly due to the unique challenges of creating recombinant Synechocystis sp. PCC 6803. We believe that this plasmid will be a great tool for fellow iGEMers working with cyanobacteria.
Source
The rest of the backbone is taken from pSB1C3 starting from annealing sites for VF2 and VR.
Slr0168 is a neutral sequence taken from Synechocystis sp. PCC 6803.
References
The counter-selection homologous recombination method was developed by Cheah, Y. E., Albers, S. C., & Peebles, C. A. (2012). A novel counter-selection method for markerless genetic modification inSynechocystissp. PCC 6803. Biotechnology Progress, 29(1), 23-30. doi:10.1002/btpr.1661.