Difference between revisions of "Part:BBa K1129006:Experience"

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

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 37°C 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 37°C. Consistent with Cas9 expression. No corresponding band found in the uninduced control.

T4 Phage infection by British Columbia iGEM 2013

FIGURE 1: Our first goal was too measure bacterial growth kinetics under T4 phage predation with our host strain and experimental conditions. The top left corner has the estimated inoculums and four different dilutions of phage were assessed. The results are consistent with a step-wise lysis cycle that is well documented in the literature. See the modeling section for more detailed analysis of the kinetics. The effect of initial T4 bacteriophage concentration on E.coli GB10 (without any CRISPR/Cas9 cassettes) cell growth for various T4 phage inoculums. Overnight cultures of wild-type E. coli 10G cells were grown at 37 °C until an OD of 0.3 was obtained diluted to each inoculum, the cells counts of which were estimate by plate counts (CFU). The cultures were grown in a 96-well format and OD was measured every 5 min for 2 hrs (n = 2). Consistent with Figure 2. Measuring OD of combinatorial library clones after incubation for 24 hours at 30 °C.The horizontal red line represents the threshold for selection of clones for sequencing and colored points correspond to characterization data in the subsequent figure. 103 phage were added to each well. Figure 3. Measuring final OD of select combinatorial library clones under different arabinose concentrations after incubation for 24 hours at 30 °C. Well IDs correspond with the precending figure. 103 phage were added to each well. Figure 4. The effect of phage predation on clones from combinatorial libraries. Clones were arrayed into a 96-well plate and grown for 6 hours at 30 °C. 103 phage were then added to each well and OD was monitored overnight. The results seem to resolve two groups, one showing increased final OD over the other (blue and red lines respectively (n=2). Figure 5. Monitoring growth of two select combinatorial library clones under high cell inoculum. 10^3 phage were then added to each well and OD was monitored overnight (n=2).