Assembly of pSB1AC3-J45320.J45180 (J45800)
pSB1AC3-J45320.J45180 prepped and submitted for sequencing. Results likely tomorrow afternoon according to sequencing center (marked as high priority).
- I prepped DNA from two colonies - 1-2(Austin's Mach 1 transformation) & 2-4(Reshma's TOP10 transformation).
- 1-2 - 190ng/μl - used 2μl for sequencing reaction
- 2-4 - 268ng/μl - used 1.2μl for sequencing reaction
- I used 4 primers for each - VF2, VR, pchA mut forward, and osmYR. The last two are mutation primers so who knows if they will work.
- I tried a colony PCR yesterday using pchA mut forward and osmYR. Only 2-4 amplified and it had multiple bands with the brightest being at 1kb. There was a weaker band at ~1900bp, the expected product length.
- 100mL cultures of these two colonies were grown up overnight. They DO NOT smell like mint this morning to either Tom or Reshma. Austin thinks there may be a whiff of mint from the Mach1 culture. Note that they are in TOP10 and Mach1 cells.
- Screening 16 more colonies by smell today. Started 10 ml cultures of 6 colonies from the TOP10 transformation and 10 from the Mach1 transformation.
Assembly of pSB1AT3-J45250.J45400 (J45600)
- Both Austin and Reshma got colonies in Mach1 and TOP10 cells.
- Letting these colonies grow up more.
- No transformants in IK cells.
- Started 8 4mL cultures of colonies. 4 from Reshma's TOP10 plates and 4 from Austin's Mach1 plate. Do smell tests, prep and digest tonight (to check if it is right).
- If someone has time to set up a colony PCR on this, let me know.
- The cotransformation of pSB1AC3-J45250 with pSB3K3-J45400 into IK cells failed.
- Transformation of pSB1AC3-J45250 into IK cells worked.
- Submitted pSB1AK3-J45180 to the Registry.
- Use 4.4μL stock IA-OH per 10mL culture
- Use 40μL 0.5M SA per 10mL culture
- 50mL of each LB-Amp. In 250 mL flask.
- Used 0.625mL of pSB1AC3-J45200 (OD600nm=0.80) in each flask
- Used 0.793mL of pSB1AT3-J45180 (OD600nm=0.63) in each flask
- Used 0.531mL of pSB1AC3-J45250 (OD600nm=0.94) in each flask
Thus, each strain was diluted back to OD 0.01 in each flask.
- Coculture pSB1AC3-J45250 and pSB1AK3-J45180 without salicylic acid and isoamyl alcohol
- Coculture pSB1AC3-J45250 and pSB1AK3-J45180 with salicylic acid and isoamyl alcohol
- Coculture pSB1AT3-J45200 and pSB1AK3-J45180 without salicylic acid and isoamyl alcohol
- Coculture pSB1AT3-J45200 and pSB1AK3-J45180 with salicylic acid and isoamyl alcohol
- pSB1AC3-J45700 <--- for possible cotransformation with J45250.J45400 -- done
- pSB3K3-J45400 <--- for possible cotransformation with J45250 -- done
- The chemically competent IK cells are not very competent.
- The electrocompetent IK cells are sufficient for intact plasmid transformation.