- This project is funded by the National Institutes of Health – National Institute of Allergy and Infectious Diseases grant – 1R24AI155395
- The cartoon shows the generic construct used to create each GC knockout mutant using synthetic DNA
- 500bp upstream/downstream: these are homologous regions outside the gene of interest that will facilitate homologous recombination events (2) to result in a insertion
- 15bp 5’/3′ of gene of interest: the 5′ end contains the start codon plus 4 more codons. The 3′ end contains 4 codons then the stop codon. It is important that your gene is in the same direction as the Kanamycin gene because you want to have the same transcriptional polarity.
- Kan Primer: the purpose of the Kan primer is that this region is what we will have our forward/reverse primers bind to when we run PCR to amplify our construct then screen using gel electrophoresis to confirm transformation
- Bar Code: this is a unique barcode for each KO locus to identify our mutants generated
- PacI Cut-Site: an 8bp sequence that will be recognized by its restriction enzyme, that when recognized will cut both strands of the DNA creating a 3 ‘ 2bp overhangs
- Kanamycin Promoter + Kanamycin Resistance Gene: selection marker
- NotI Cut-Site: an 8bp sequence that will be recognized by its restriction enzyme, that when recognized will cut both strands of the DNA creating a 5′ 4bp overhangs
- Cut Sites: cut sites on either end of the Kanamycin cassette allow you to insert new cassettes between the two cut sites.
- 12bp DUS site: the 12bp DNA Uptake Sequence is present to ensure the GC take up the DNA efficiently
- Kanamycin Cassette sequences are found here
- We supply chromosomal DNA of each successfully transformed mutant
- For FA1090 mutants, N-1-60 is the parent strain which is an FA1090 multisite G4 mutant, 1-81-S2 pilE variant, pilC2PL ON – click link for reference paper. The official GenBank link is found here
- For MS11 mutants, HL-1-22 is the parent strain which the GenBank link is found here
- Fasta Files for sequences here
A general protocol for when your gDNA of interest arrives can be found here