3. Transformation

Transformation

Transform 5µL of dialyzed ligation into 85µL of '5X' concentrated E. coli WM6026 electrocompetent cells. Select on LB, 300 µM DAP, 100 µg/mL carbenicillin 2% agar 150 mm plates. Incubate for 12-18 hours at 37°C.

E. coli library in E. coli host

Figure 1: E. coli transposon library in E. coli host. 3.6 million colonies across 30 plates. Scraped and resuspended into aliquots to create sJMP5137.

E. cloacae library in E. coli host

Figure 2: E. cloacae transposon library in E. coli host. 2.4 million colonies across 30 plates. Scraped and resuspended into aliquots to create sJMP5138.

K. pneumoniae in E. coli host

Figure 3: K. pneumoniae transposon library in E. coli host. 1.5 million colonies across 30 plates. Scraped and resuspended into aliquots to create sJMP5139.

Cell Collection and Storage

1. Add approximately 15mL LB + 300µM DAP to a plate.
2. Use a plate scraper to remove colonies from the agar surface.
3. Gently stir and scrape to resuspend cells in media.
4. With a 10 mL pipette, transfer the liquid to another plate and repeat scraping and resuspension.
5. Transfer resuspended cells to a sterile container on ice.
6. Repeat the process until all plates have been scraped.
7. For storage, prepare a glycerol stock of the Mobile-CRISPRi pooled library.
   • Normalize the OD600 to approximately 10-30.
   • Add sterile glycerol to a final concentration of 15%.
8. Aliquot into cryovials and store at -80°C.