IGEM:MIT/2006/Notebook/2006-8-11

To do

 * 1) Recontact Jose Aleman in Stephanopoulos lab. I (austin) talked to him today. He said they fixed their GC yesterday.
 * 2) smell overnight IK smell testers and after making glycerols from regular batch then add precursor to the daytime smell testers -done
 * 3) analyze sequencing from weds [full assemblies] veena and kate, afternoon
 * 4) List Biobrick Molecules
 * 5) add pchBA to registry
 * 6) *Lets update our index with our numbering system and all the biobrick constructs we believe that we have (and then we can check them off as we get the sequence results back. Also, BO could you put in links to each part's registry page on the index!)
 * 7) digest WGD with EP and run on a gel with 8/10 pUCP22:EP digest digest started 12:30
 * 8) ligate WGD into EP cut pUCP22 shuttle vector and transform into Top10 in the pm
 * 9) autoclave waste
 * 10) digest pCHBA with E and S and backbone with E and S and ligate digest started 12:30
 * 11) mutagenesis of BAT2, transformation of BAT2, ATF1, and THI3 mutagenesis Bo and Stephen pm
 * 12) LCs of R0040.E0840 and osmY.E0840s
 * 13) GC of smell tests decided to run it when we have more controlled samples to run
 * 14) miniprep RBS.coding.terminators to inverters digests of these started at 12:30

Experiment description

 * We would like to know the amount of time it takes to produce the final volatile compound.
 * I added SA (2mM) and IA-OH (10mM) to 5mL pre-grown Indole KO cultures.
 * Cultures containing J45100 and J45220 were further incubated at room temperature (25C)
 * Cultures containing J45120, J45170, and J45200 were further incubated (with vigorous shaking) at 37C
 * After 240 min (4hrs), I continued the experiment with just the J45200 in order to determine the amount of time it took the cells to stop producing the scent (either because the cells have reached stationary phase, started dying, etc.)

Conclusions

 * osmY is probably not correctly assembled in the BioBrick (J45140).
 * Some smell is obtained from cells that are incubated at 25C, indicating that ATF1 protein is present in a sufficient amount after overnight culturing.
 * Smell is obtained much more quickly when incubation is resumed at 37C as opposed to 25C. This is likely because the ATF1 protein present in the overnight culture is supplemented by increased protein production at 37C.
 * Because smell (banana) was detected after only 15 min of incubation at 37C (J45200), this means that sufficient IA-OH enters the cells within 15 minutes of addition.

ligations completed (Kate and Veena)

 * 1) pUCP22 [EP] + J45100 [EP] ->amp/gent
 * 2) pUCP22 [EP] + J45120 [EP] ->amp/gent
 * 3) pchBA [ES] + pSB1AK3 [ES] ->amp/kan
 * 4) pchBA [ES] + pSB1A3-1 [ES] ->amp
 * 5) Q04400 [ES] + J45099 [XP] + pSB1AT3 [EP] ->amp/tet
 * 6) Q04400 [ES] + J45119 [XP] + pSB1AT3 [EP] ->amp/tet
 * 7) Q04400 [ES] + J45199 [XP] + pSB1AT3 [EP] ->amp/tet
 * 8) Q04400 [ES] + J45219 [XP] + pSB1AT3 [EP] ->amp/tet
 * 9) osmY annealed [ES] + E0840 [XP] + pSB1AT3 [EP] ->amp/tet