CRISPR Interference-Based Gene Silencing: A Technique for Targeted Repression of Gene Function in Pathogenic Leptospira
Transcrição
For conjugation, Leptospira cells are grown at 29 or 37 degrees Celsius in HAN media. One day before conjugation, donor E. coli cells are grown in LB media supplemented with Diaminopimelic acid – DAP, and spectinomycin.
Next, in the day of conjugation, grow the saturated E. coli cultures in LB plus DAP. Inside a biosafety hood, assemble the filtration apparatus by placing the membrane filter on top of the glass base, followed by 15-mL glass funnel. Both pieces are held together by the spring clamp. Then, connect the glass to a vacuum pump. Add 5 ml of Leptospira cultures to the funnel.
Next, add the E. coli β2163 strain containing the plasmid of interest. The volume will vary according to the optical density of the culture. We are aiming to 1:1 cell proportion. Turn the vacuum pump. Now, liquids will get through the membrane, and cells will be concentrated on top of it.
Take the filter, and place it onto EMJH plate plus DAP, bacterial side up. Incubate the plates at 29 degrees Celsius for 24 hours. Then, recover the filters from the plates, and place it into 50-mL conical tube.
Use 1 mL of liquid HAN media to recover the cells from the filter by pipetting. Visualize the recovered cells by dark-field microscopy.
At this stage, equivalent numbers of E. coli and Leptospires can be seen. 100 to 200 microliters are used to spread the bacteria on two HAN plates containing spectinomycin. At this stage, DAP is omitted, and E. coli will not grow. Plates are incubated at 37 degrees Celsius and 3% CO2. Colonies should be apparent between 8 to 10 days.
