Difference between revisions of "Part:BBa K1218011:Experience"

CU-Boulder 2014

BBa_K1218011 can be targeted to a DNA sequence through the modification of its spacer region.

A 30mer spacer sequence targeting the neomycin phosphotransferase gene was designed and substituted for the original spacer in BBa_K1218011. BW23115 E coli with the neomycin phosphotransferase inserted into the genome were chemically transformed with the original and modified BBa_K1218011 to compare CRISPR-Cas9 specificity. Transformants were selected for on chloramphenicol.

Figure 1: Transformation results of neomycin resistant E. coli with Cas9 part having either A) non-targeting or B) targeting spacer sequence.

There was a substantial decrease in growth between the non-targeting (1920 colonies) and the targeting sample (8 colonies) that must be accredited to the differences in spacer sequence. As can be seen in Figure 1: B, there is growth in the targeting sample. Sequencing showed that all eight colonies had deleted the spacer region and one or both of the adjacent repeats.

Part BBa_K1218011 was further improved by the addition of the M13 packaging signal (BBa_K1445000) to form the composite part BBa_K1445001. This part was packaged into phage and introduced via infection to conjugated neomycin resistant bW23115 E. coli. Sample was plated on chloramphenicol to select for cells infected with the phage delivering the phagemid with the Cas9 part and M13 origin of replication.

Figure 2: Infection results of BBa_K1445001 on a pSB1C3 backbone, grown on LB agar with 170 ug/mL chloramphenicol.

The growth in figure 2 demonstrates that, with the addition of the M13 origin of replication, BBa_K1218011 can be delivered to cells via recombinant M13 phage.