The CyanoSource library is a standardised resource of 1) single gene, barcoded knockout mutants for the glucose tolerant strain of the model cyanobacterium Synechocystis sp. PCC 6803 and 2) the plasmids to generate the mutants. The library was developed as a collaboration between the University of East Anglia and Earlham Biofoundry in Norwich, and the University of Edinburgh and the the Edinburgh Genome Foundry in Edinburgh. The library allows functional studies on a genomic level, while gene specific barcodes offer a tool to determine the individual fitness of mutants in pooled cultures.
In each mutant the complete or partial coding sequence of individual genes has been replaced with an insertion sequence containing a gene specific barcode, a kanamycin resistance selection cassette (KmR) expressing the aph(3')-Ia gene, and an unmarking cassette consisting of either the sacB gene from Bacillus subtilis encoding levansucrase (glycosyltransferase) (Lea-Smith et al., 2016 J Vis Exp 111: e54001), or a genetically modified cytosine deaminase (codA) gene (Young et al., 2014 Plant Journal 80: 915). The cyanobacterial mutants are either fully segregated when possible (e.g. for nonessential genes) or partially segregated (e.g. for essential genes). Please use Search to find genes of interest).
llustration of marked mutant generation:
The plasmids are designed to allow editing of the insertion sequence region for subsequent re-transformations (e.g. to unmark the loci of interest or re-introduce modified gene variants).
Illustration of unmarked mutant generation:
Each plasmid contains the barcode (18 bp), selection cassettes (sacB-KmR or CodA-KmR) flanked with three type II restriction endonucleases sites (BpiI, AarI and SapI), a 'Left Flank' sequence homologous to the region upstream of the target gene and a 'Right Flank' sequence homologous to the region downstream of the target gene. Please see Design for more details.
• Fully segregated or are partially segregated gene deletion mutant strains for each of the 3,568 genes (see Search - please note, this work is ongoing).
• The gene sequence is replaced with an insertion sequence carrying a barcode, a kanR cassette for positive, and a codA or sacB cassette for negative selection.
• The transformation plasmid for each gene contains flank sequences (each 400 bp, but longer in some cases) and the insertion sequence (see Design).
• The plasmid can be modified and re-transformed into WT 6803 or the mutant strain to replace the insertion sequence with a sequence of choice.
The work is funded by the BBSRC.