Part:BBa_K1129008:Experience

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Applications of BBa_K1129008

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are loci found in some bacterial and archaeal genomes that, together with associated Cas (CRISPR-associated) genes function as an adaptive immune system in prokaryotes. While the details of immunity-conferring “spacer” sequence acquisition are still being worked out, it is known that exogenous DNA is processed by Cas proteins into short (~30 base) sequences that are adjacent to the Protospacer Adjacent Motif (PAM) site. These short pieces of DNA are then incorporated into the host genome between repeat sequences to form the spacer elements. The repeat-spacer-repeat array is constitutively expressed (pre-CRISPR RNAs, pre-crRNAs) and processed by Cas proteins to form small RNAs (crRNAs). The small RNAs are then loaded into Cas proteins and guide them to initiate the sequence-specific cleavage of the target sequence (or the protospacer).

Data

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).

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