IGEM:MIT/2006/Notebook/2006-10-31

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Overnights

pSB1AC3-J45250/pSB3K3-J45400

pSB1AC3-J45250/pSB3K3-J45400 didn't grow.

pSB1AT3-J45700

Austin's pSB1AC3-J45700 didn't grow in Amp/Cm but did in Amp. Reshma's did (some).

pSB1AT3-J45400

  • Amp/Tet liquid culture didn't grow
  • did grow on amp plate
  • took colonies and innoculated into amp and amp/test media --Austin Che 11:29, 31 October 2006 (EST)

Austin's to do list

  • Miniprep cultures
    • Available cultures: 250, 320, 700
  • Digests:
    • ES: 250, 320, 700
    • XP: 180, 250, 400
    • EP plasmids: 2K4, 1AC3, 1AK3
  • Ligations:
    1. 1AC.320.180
    2. 2K|1AK.250.400
    3. 2K|1AK.700.250
    4. 2K|1AK.700.400
  • Transform into MachI
  • Pick J45180 colonies to grow o/n to glycerol
  • Send J45180 plate to registry

Reshma's assemblies

To make

  • pSB1AC3-J45320.J45180
  • pSB1AK3-J45250.J45400
  • pSB1AK3-J45250.J45700
  • pSB1AK3-J45700.J45400

Step 1

  • Cut J45250 with ES - done
  • Cut J45700 with ES - done
  • Cut J45700 with XP - done
  • Cut pSB1AK3-P1010 with EP - done
  • Cut pSB1AC3-P1010 with EP - done

Step 2

  • Purify ES cut J45250 - done
  • Purify ES cut J45700 - done
  • Purify XP cut J45700 - done
  • Purify ES cut J45320 - done
  • Purify XP cut J45180 - done
  • Purify XP cut J45400 - done
  • Phosphatase treat pSB1AK3-P1010 - done
  • Phosphatase treat pSB1AC3-P1010 - done

Step 3

  • Purify pSB1AK3-P1010 - done
  • Purify pSB1AC3-P1010 - done

Step 4

Ligations: 2μL vector; 4μL each insert, 10μL quick ligase buffer

  • pSB1AC3-J45320.J45180 - used 1μL quick ligase
  • pSB1AK3-J45250.J45400 - used 1μL quick ligase
  • pSB1AK3-J45250.J45700 - used 1μL T4 ligase
  • pSB1AK3-J45700.J45400 - used 1μL T4 ligase

Incubated at RT for 5 mins.

Step 5

Transformations into Invitrogen TOP10 cells Heat shock 1min.

Smell tests

  • Use 4.4μL stock IA-OH per 10mL culture
  • Use 40μL 0.5M SA per 10mL culture

These were started today

  • 100mL of each LB-Amp. In 500mL or 1L flask.

Dilutions

  • Used 1mL of pSB1AC3-J45200 (OD600nm=0.31) in each flask
  • Used 0.27mL of pSB1AT3-J45120 (OD600nm=1.11) in each flask
  • Used 0.27mL of pSB1AC3-J45250 (OD600nm=1.11) in each flask

Thus, each strain was diluted back to OD 0.003 in each flask.

Samples

  1. Coculture pSB1AC3-J45250 and pSB1AT3-J45120 without salicylic acid and isoamyl alcohol
  2. Coculture pSB1AC3-J45250 and pSB1AT3-J45120 with salicylic acid and isoamyl alcohol
  3. Coculture pSB1AT3-J45200 and pSB1AT3-J45120 without salicylic acid and isoamyl alcohol
  4. Coculture pSB1AT3-J45200 and pSB1AT3-J45120 with salicylic acid and isoamyl alcohol
  5. Culture pSB1AC3-J45250 without salicylic acid and isoamyl alcohol
  6. Culture pSB1AC3-J45250 with salicylic acid and isoamyl alcohol
  7. Culture pSB1AT3-J45200 without salicylic acid and isoamyl alcohol
  8. Culture pSB1AT3-J45200 with salicylic acid and isoamyl alcohol

Results

  • J45200 has some smell in the presence of precursors but J45250 does not during exponential phase.
  • Coculture of pSB1AC3-J45200 and pSB1AT3-J45120 in the presence of precursors smells minty in exponential phase.
  • Coculture pSB1AC3-J45250 and pSB1AT3-J45120 in the presence of precursors maybe smells minty in exponential phase.
  • Coculture of pSB1AC3-J45200 and pSB1AT3-J45120 in the presence of precursors smells like banana in stationary phase.
  • Coculture pSB1AC3-J45250 and pSB1AT3-J45120 in the presence of precursors smells like banana in stationary phase. You could maybe argue that this one smells more like banana than the coculture of pSB1AC3-J45200 and pSB1AT3-J45120.
  • Both J45200 and J45250 smell like banana in stationary phase.

These still need to be done

  1. Coculture pSB1AC3-J45250 and pSB1AK3-J45180 without salicylic acid and isoamyl alcohol
    • Couldn't start. No pSB1AK3-J45180.
  2. Coculture pSB1AC3-J45250 and pSB1AK3-J45180 with salicylic acid and isoamyl alcohol
    • Couldn't start. No pSB1AK3-J45180.
  3. Coculture pSB1AC3-J45250/pSB3K3-J45400 and pSB1AT3-J45700 without salicylic acid and isoamyl alcohol
    • Couldn't start. No pSB1AC3-J45250/pSB3K3-J45400.
  4. Coculture pSB1AC3-J45250/pSB3K3-J45400 and pSB1AT3-J45700 with salicylic acid and isoamyl alcohol
    • Couldn't start. No pSB1AC3-J45250/pSB3K3-J45400.
  5. Coculture pSB1AT3-J45200 and pSB1AK3-J45180 without salicylic acid and isoamyl alcohol
    • Couldn't start. No pSB1AK3-J45180.
  6. Coculture pSB1AT3-J45200 and pSB1AK3-J45180 with salicylic acid and isoamyl alcohol
    • Couldn't start. No pSB1AK3-J45180.

Yeast Stuff

  1. Grew up overnights of ADH1promoter+BSGD in pRS306.
    1. Oddly enough, only 3 of my 9 overnights grew up. Those 3 had been innoculated with a ton of cells.
  2. Miniprepped vectors and digested to linearize.
    1. All enzymes linearized the vectors beautifully, except NdeI only partially digested G5 miniprep.
    2. Thus, combine NcoI, NdeI, StuI, and XcmI digests into three master tubes (one each for G5, G6, G7), and transform on Nov. 1, after SB lunch.
  3. Yeast cells didn't grow up overnight.
    1. This was unexpected, as a parallel culture grown overnight in the same media grew fine. I can only conclude that the slow growth was due to the fact that I innoculated from a freezer stock, which I have never done before and so don't know what to expect.
    2. I will transform the yeast tomorrow (Wed), and cut even more corners to get a culture ready for Friday.

General to do list

  • Streak and start overnight culture of pSB1AC3-J45250/pSB3K3-J45400 again.
  • Cotransform pSB1AC3-J45250 and pSB3K3-J45400 again into IK cells
  • Make more LB media
  • Start overnight cultures of
    • pSB1AT3-J45120 (grows fast)
    • pSB1AK3-J45180 (do extra just in case)
    • pSB1AC3-J45250 (grows fast)
    • pSB1AT3-J45200 (do extra, grows slow)
    • pSB1AC3-J45700 (do extra, grows slow)
  • Autoclave some 250 mL flasks covered in foil - done
  • Start overnight of IK cells to make competent cells tomorrow

Parts missing from the Registry

  • BBa_J45009
  • BBa_J45300
  • BBa_J45992
  • BBa_J45993
  • BBa_J45398
  • BBa_J45995
  • BBa_J45996

Done

pSB1AT3-J45400 submitted to Registry.

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