WhiteFeather participated in a DIY bio CRISPR CAS-9 workshop hosted by BricoBio and Fluxmedia. The project undertaken with several BricoBio/Fluxmedia members was to use the CRISPR kit to transform E. coli to be resistant to two types of antibiotics. The kit was assembled by and acquired from Josiah Zayner.
The Protocol/ Experiment undertaken at BricoBio
Step 1: fill 4 microcentrifuge tubes w/ 100uL buffer + 6 colonies each –
Tube 1 – HME63 E. coli strain (CRISPR kit), from filter paper sample plate
Tube 2 – HME63 E. coli strain (CRISPR kit), experiment plate
Tube 3 – OP50 E. coli strain (BricoBio source kit)
Tube 4 – HME63 E. coli (CRISPR kit), from filter paper sample plate
Step 2: add plasmids to tubes –
Tube 1 – Cas9/ tracrRNA (accidentally put 20uL instead of the required 10uL) + rpSL Template DNA 10uL + crRNA 10uL
Tube 2 – Cas9/ tracrRNA 10uL + rpSL Template DNA 10uL
Tube 3 – rpSL Template DNS 10uL + crRNA 10uL
Tube 4 – crRNA 10uL
*technically only Tube 1 should produce the desired transformation – all three others would be surprise results (if any results).
Step 3: put all samples in fridge x 30mins
Step 4: heat shock all samples @42˚C x 30sec + then put back on cold block
Step 5: 100uL/ each of 4 samples into LB broth in separate microcentrifuge tubes
Step 6: add the 4 filled tubes to 37˚C warm water bath x 1hr
Step 7: sterilize inoculation loops in EtOH and streak 100uL of each sample (in tubes) onto agar plates
Step 8: let streaked plates sit x 10mins, turn over and leave O/N at RT or incubate at 37˚C
Results after three days: Unsuccessful!
Possible reasons, described by a BricoBio group member:
The current hypothesis is that our cells are not competent, and are not picking up the plasmids. There are a number of possible causes to this problem, and a number of potential solutions. I’m not sure if the cells were stored in the transformation mixture overnight, after being picked off the plates, and before they were used. If they weren’t, this could have been the issue. (Side note: They weren’t.) After having plated the cells on Saturday, I should have perhaps come in Sunday evening to store the cells in the transformation buffer. We don’t have all the required equipment though, like the 37 degrees agitator or the -80 freezer. We could either try and work with the equipment we have, or prepare the cells in a better equipped lab beforehand. Highly competent cells are also available commercially. It is expensive though, and goes against the ethos of DIY molecular biology. If we can solve our issues with making competent cells, we should be able to carry out more successful transformations, hopefully.