Part:BBa_K3044016:Design
Conjugative sgRNA-dCas9 system
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 478
Illegal BglII site found at 1552 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4547
Illegal NgoMIV site found at 4557 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 4449
Design Notes
During the optimization illegal restriction sites were avoided. Another thing to keep in mind was to figure out which codons to choose for E. coli K12.
The sgRNA was designed according to the design protocol "CRISPR interference (CRISPRi) for sequence-specific control of gene expression". [1] An important thing to keep ind mind when designing a sgRNA is off-targets. Therefore, the sgRNA sequence was aligned to the core genome of our chassis E coli K12 TOP10 and KG22 and no off-targets were found. Though, off-targets may appear in other bacteria strains.
The location of the OriT in the plasmid had to be concerned as well and was therefore implemented behind part BBa_K3044020 to ensure correct delivery of the sgRNA-dCas9 system to the target bacteria.
Source
The original sequence of the dCas9 is from BBa_K1150000 and was codon optimized based on a codon frequency table [2]
The sequence of the sgRNA is designed on the basis of the GFP sequence. The target sequence is determined according to the location of PAM sequences in the target sequence, which the dCas9 protein recognizes.