IGEM:MIT/2006/Notebook/2006-11-2

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/&mu;l - used 2&mu;l for sequencing reaction
 * 2-4 - 268ng/&mu;l - used 1.2&mu;l 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.

Other transformations

 * The cotransformation of pSB1AC3-J45250 with pSB3K3-J45400 into IK cells failed.
 * Transformation of pSB1AC3-J45250 into IK cells worked.

Registry

 * Submitted pSB1AK3-J45180 to the Registry.

Smell tests

 * Use 4.4&mu;L stock IA-OH per 10mL culture
 * Use 40&mu;L 0.5M SA per 10mL culture


 * 50mL of each LB-Amp. In 250 mL flask.

Dilutions

 * 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.

 
 * 1) Coculture pSB1AC3-J45250 and pSB1AK3-J45180 without salicylic acid and isoamyl alcohol
 * 2) Coculture pSB1AC3-J45250 and pSB1AK3-J45180 with salicylic acid and isoamyl alcohol
 * 3) Coculture pSB1AT3-J45200 and pSB1AK3-J45180 without salicylic acid and isoamyl alcohol
 * 4) Coculture pSB1AT3-J45200 and pSB1AK3-J45180 with salicylic acid and isoamyl alcohol

Miniprep

 * pSB1AC3-J45700 <--- for possible cotransformation with J45250.J45400 -- done
 * pSB3K3-J45400 <--- for possible cotransformation with J45250 -- done

Competent cells

 * The chemically competent IK cells are not very competent.
 * The electrocompetent IK cells are sufficient for intact plasmid transformation.