HacDC CRISPR

Project Description

CRISPR-Cas9 is a groundbreaking new gene editing technique in molecular biology. In brief, ten years ago it was found that bacteria possess a self-defense mechanism against viruses that includes a virus definition database and a gene removal mechanism. The bacterial chromosomal DNA contains small pieces of the DNA sequences of harmful viruses. The defense mechanism reads the chromosomal DNA and compares it to the known virus sequence. If a match is found, the sequence is excised. This mechanism can therefore be exploited to excise any gene by replacing the virus definition sequences in the CRISPR sequence with the target sequence. Once the DNA is cut, a new gene can be inserted with reasonable success. The cost of this technique is dramatically reduced from pre-existing gene editing techniques.

Read more here: https://en.wikipedia.org/wiki/CRISPR

In the ODIN kit experiment, bacteria (E. coli HME63 strain) are modified to add resistance to the antibiotic streptomycin. The kit provides the vulnerable bacteria, the resistance gene, and growth media with and without antibiotic. The original unmodified bacteria can only grow on the plain agar media whereas bacteria with a successfully edited genome will also grow on the streptomycin-laced agar.

Activities and Goals

DC CRISPR Initiative is our effort to learn about, perform, and teach CRISPR genetic editing at HacDC. To begin the project, we’ve ordered a Do-It-Yourself CRISPR Kit, which includes (supposedly) all the tools and ingredients needed to perform a CRISPR procedure a few times. We’ll hold a few events at HacDC to go through the procedure and document our experience. Eventually we’ll create a guide that older high school kids can follow. This project also explores interest in molecular biology and genetics at HacDC. We're just starting! Keep an eye out for CRISPR events in our MeetUp page, on the mailing list, and our Blabber discussion forum.

Project Team Members

Enrique C. - Project Manager and Point of Contact Nancy W. - Project Development Lead

Worklog

July 30, 2016 We received the CRISPR kit purchased with Project EXPANSION funds (thanks!).

August 2, 2016 Nancy and Enrique inventoried the ODIN kit and designated the small classroom fridge as the "NO FOOD" Project CRISPR fridge.

August 5, 2016 Nancy and Enrique prepared four Petri dishes (two plain agar, two streptomycin-agar). The agar and antibiotic(streptomycin)-laced agar are gel-like substances similar to gelatin. They come as powders which must be mixed with water and heated to dissolve. The recipe is proportioned for seven Petri dishes but we scaled down to one of each, scaling the agar powders and water by one-seventh. Even so we were able to coat two dishes with each growth medium. We didn't have distilled or deionized water and used bottled purified drinking water in a pinch. The mixture (agar gel only, no bacteria!) was heated in the microwave 7 seconds at a time. It took 4-5 cycles until the powders were fully dissolved and the liquid transparent, then another 5 minutes until they were cool enough to handle and pour into the plastic Petri dishes. The dishes cooled at room temperature for an hour to remove some condensation (the covered hot liquid creates condensation on the lid), then placed in the fridge. Two are agar (no antibiotic) and two are streptomycin/Kan agar (antibiotic laced).

August 10, 2016 Ken, Bobby, Nancy, and Enrique. We streaked some of the original E. coli HME63 bacteria onto two plain agar plates. Plate 1 was left out tonight (the bacteria need to grow). Plate 2 was immediately refrigerated and will be taken out to grow just before the actual experiment.