IGEM:MIT/2006/Notebook/2006-9-5

Meeting notes!

 * 1) BGD/WGD
 * 2) *graphs (GC)
 * 3) IAOHGD
 * 4) *THI3 mutagenesis (done?)
 * 5) *ATF1 silent mutagenesis (sequence is back)
 * 6) *construction:
 * 7) **3.B.3.THI3 assembled, sequence correct
 * 8) **3.B.3.THI3-mut [398] 9/5: cut with SP, ran on gel, lane 9 looked right and lanes 1 and 2 were possibly right
 * 9) **3.B.3.THI3-mut.15 [399] 9/5: ligated SP cut & pcr cleaned up 3.B.3.THI3-mut with XP cut B0015, and transformed; backbone was A/C. the most likely to work is the one using the 3.B.3.THI3-mut from lane 9...so, liquid culture like 5 of the colonies from that plate (it's ligation 1 on the ligation sheet from 9/5)
 * 10) **R0011.3.B.3.THI3-mut.15 [400]
 * 11) SAGD
 * 12) *construction:
 * 13) **pchBA-mut.15 [298] assembled, sequence correct
 * 14) **RBS.pchBA-mut.15 [319] assembled, sequence correct
 * 15) **R0011.RBS.pchBA-mut.15 [320]
 * 16) *PCR pmsCEAB, run a gel the "R" pcr seemed to work...what exactly does that mean, Kate? --okay this is good. i thought that R (as in pE3R) was most likely to work because the other two minipreps prob failed for whatever reason (really low concentrations). so this PCR piece is complete with Promoter, Rbs, and Term. The next step is to cut it and put pmsCEAB into a double-antibiotic backbone for a double selective transformation. we must watch out for Pst sites though!! and we should make sure that we use a diff backbone than our WGD devices have
 * 17) pseudomonas
 * 18) *transform
 * 19) *biofilter research
 * 20) yeast
 * 21) *pellet big culture -done
 * 22) *bake w/o BGD
 * 23) *miniprep 2 YCP vectors
 * 24) *digest YCP vectors with XS -> freeze step

Update
1. Digested the 10 hopefully mutagenized 30.BAT2.30.THI3 structures and ran on gel.
 * I am fairly confident that digest 9 worked. Digest 1 and 2 may have worked.  Thus, I will PCR cleanup all three.

2. Ran the PMS PCR products on a gel and found that the only one with the correct band is the R tube. I will PCR cleanup that and put it into the metal blue holder labeled "PMSR cleaned up."

3. I am assuming that wells E10, E11, and E12 are the osmY.inverter.e0840 structures. I would say that they were moderately successful. At around stationary phase, fluorescence measurements on those wells leveled off, suggesting that no more GFP was being made in stationary phase^. However, for some reason overall GFP production was roughly twice as large as it was for the R0040 and osmY structures. We may want to keep an eye on this, but it seems as if we should move on and see how the osmY.inverter.xxxs and osmY.xxxs work with the GC. Some time next week, if I can be shown exactly what glycerols to use, I can redo everything at standard conditions (Jason gave me some hints). I will do samples of osmY.e0840 (Short and Long), R0040.e0840, TOP 10, and osmY.Inverter.e0840. Also, I need to know if the osmY.e0840s are on different backbones than the osmY.inverter.e0840s since Jason said this may make a difference. It may explain why there is twice as much GFP production in the osmy.inverter.e0840 structures.

^This actually makes a lot of sense since GFP is a very, very stable protein.

okay stephen: so all the sequence confirmed constructs that you are looking for are in the final biobrick''' box. you should look specifically in the cluster of 4 tubes in a square in one corner, in the line of 3 alone in the middle of one side, and in the third major row of all the longish rows running right above the top-labeled biobrick constructs. you will find everything you mentioned here. labels are decent. hopefully the antibiotic is written on each glycerol. if not, check the sequence order or the LCs i used are still in fridge with labels. good luck. if you have trouble, i can easily find them for you, but hopefully this helps. if you want me to put them in a seperate box next time i go to lab (9/6 pm) that would work too. just let me know'''