Part:BBa_K1129006:Experience

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Applications of BBa_K1129006

The Cas9 construct was transformed into E. cloni 10G, plated on LB agar plates made with chloramphenicol (33ug/mL), and grown overnight at 37C.

A single colony was picked and grown overnight in LB (with chloramphenicol) at 30C and shaking (1000 rpm). Growth was very slow for the Cas9 culture and OD was only ~0.6 in the morning. Several aliquots of 175 uL were taken and placed in separate wells of a 96-well plate. They were induced with varying concentrations of arabinose (0, 0.5%, 1%, and 2%) for three hours at 37C and 1000 rpm.

OD was measured and it was determined that there was a tradeoff between induction concentration and growth. The highest induction concentration that also had relatively good growth was 1% arabinose (OD=1.17 vs 0.98 for 2% induction and 1.67 for non-induced). This culture and the non-induced control was collected, centrifuged, resuspended in SDS-PAGE loading buffer, and boiled for 10 minutes. They were loaded in a 12% gel and run at 150V for 1 hour and 10 minutes.

The gels were stained with InstantBlue overnight and destained with water for several hours.

There is a large, dark band in the Cas9 lane at the approximate size of Cas9 (163 kDa) that is not present in the uninduced control (Figure 1). This would be consistent with the successful expression of the Cas9 protein.

FIGURE 1: 12% SDS-PAGE gel showing strong expression of a protein of approximately 163 kDa in the sample that was induced with 1% arabinose for three hours at 37C. Consistent with Cas9 expression. No corresponding band found in the uninduced control.

https://static.igem.org/mediawiki/2013/0/0a/UBC-Growth_curves_MOI.png

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