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

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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.

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 4.4μL stock IA-OH per 10mL culture
  • Use 40μL 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.
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