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

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:
 * 1AC.320.180
 * 2K|1AK.250.400
 * 2K|1AK.700.250
 * 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: 2&mu;L vector; 4&mu;L each insert, 10&mu;L quick ligase buffer


 * pSB1AC3-J45320.J45180 - used 1&mu;L quick ligase
 * pSB1AK3-J45250.J45400 - used 1&mu;L quick ligase
 * pSB1AK3-J45250.J45700 - used 1&mu;L T4 ligase
 * pSB1AK3-J45700.J45400 - used 1&mu;L T4 ligase

Incubated at RT for 5 mins.

Step 5
Transformations into Invitrogen TOP10 cells Heat shock 1min.

Smell tests

 * Use 4.4&mu;L stock IA-OH per 10mL culture
 * Use 40&mu;L 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
 * 2) * Couldn't start. No pSB1AK3-J45180.
 * 3) Coculture pSB1AC3-J45250 and pSB1AK3-J45180 with salicylic acid and isoamyl alcohol
 * 4) * Couldn't start. No pSB1AK3-J45180.
 * 5) Coculture pSB1AC3-J45250/pSB3K3-J45400 and pSB1AT3-J45700 without salicylic acid and isoamyl alcohol
 * 6) * Couldn't start. No pSB1AC3-J45250/pSB3K3-J45400.
 * 7) Coculture pSB1AC3-J45250/pSB3K3-J45400 and pSB1AT3-J45700 with salicylic acid and isoamyl alcohol
 * 8) * Couldn't start. No pSB1AC3-J45250/pSB3K3-J45400.
 * 9) Coculture pSB1AT3-J45200 and pSB1AK3-J45180 without salicylic acid and isoamyl alcohol
 * 10) * Couldn't start. No pSB1AK3-J45180.
 * 11) Coculture pSB1AT3-J45200 and pSB1AK3-J45180 with salicylic acid and isoamyl alcohol
 * 12) * Couldn't start. No pSB1AK3-J45180.

Yeast Stuff

 * 1) Grew up overnights of ADH1promoter+BSGD in pRS306.
 * 2) Oddly enough, only 3 of my 9 overnights grew up. Those 3 had been innoculated with a ton of cells.
 * 3) Miniprepped vectors and digested to linearize.
 * 4) All enzymes linearized the vectors beautifully, except NdeI only partially digested G5 miniprep.
 * 5) 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.
 * 6) Yeast cells didn't grow up overnight.
 * 7) 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.
 * 8) 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.