"

From 2013.igem.org

iGEM Home

Combinatorial CRISPR array assembly and screening

Digest and ligate components to be assembled

A strategy needs to be designed such that the tracrRNA and Cas9 protein flank randomly inserted spacer regions. Here is an example of what we did:

Digest BioBrick containing [http://parts.igem.org/Part:BBa_K1129006 Cas9] and promoter with EcoRI and PstI, spacer and promoter with Xbal (ex. BBa_K1129006) and tracrRNA and promoter with Xbal1 and Pst1 (ex. BBa_K1129008, BBa_K1129009).

Run the digestion products on a 1.5% agarose gel and gel purify the digest products.

Cas9 and the spacer constructs are first ligated overnight.

A subsequent ligation is performed that adds the tracrRNA-pSB1C3 digestion products and the ligation is continued for 3 hours.

Array clones and screen for growth

The reaction is then transformed and 768 clones are arrayed into 384-well plate containing LB and chloramphenicol using a q-pix2 (Genetix) colony picking robot.

A biostack plate filler and stacker then adds 10 µg/mL of arabinose to each well and the plates are incubated at 30 °C for 24 hours.

OD is then read on a Varioskan (Thermo-Fisher) plate reader.

A visualized experiment showing all the robots and assay equipment used can be found [http://www.jove.com/video/2461/a-high-throughput-screen-for-biomining-cellulase-activity-from here]