"
Team:Hawaii/Tri-parental conjugation between E. coli and PCC6803
From 2008.igem.org
Protocol from Callahan Lab
- Based on Anabena experiments
- Grow cyanobacteria into exponential phase in liquid BG-11.
- Streak out fresh plates of transformed E. coli one day before. Use to innoculate 40 ml SOB and allow E. coli to grow until OD600=0.6.
- Pellet E. coli. Wash with 40 mL BG-11. Pellet and wash again.
- Resuspend pellet in 1.4 ml BG-11.
- Spot E. coli and cyanobacteria together on BG-11 + 5% LB plate. Use 5 μl of each E. coli strain and 10 μl of cyanobacteria.
- Wait for spot to dry on plate.
- Incubate for 2 days in light and CO2 at 30C.
- Use a loop to scrape the growth on the BG-11 + 5% LB plate.
- Streak innoculate on BG-11+sp+sm.
- Incubate in light and CO2 at 30C.
Note: Because E. coli may curtail the growth of cyanobacteria (sometimes even killing them), it is suggested that 1/1, 1/10, and 1/100 dilutions of E. coli cultures be made before spoting with cyanobacteria.
