OD600s for J45700, J45800, and MG1655
EZ + Glycerol
After 17 hours
After 22 hours
M9 Minimal + Casamino Acids + Fructose
After 18 hours
After 23 hours
M9 Minimal + Alanine, Aspartic Acid, Leucine, Proline, Serine + Fructose
After 14 hours
After 19 hours
- We will ditch M9 and move to EZ. Also, Professor Tribe said that he used Neidhardt (EZ) or 56 mineral salts to make chorismate overproducers in a quick and dirty fashion. So, our best bet seems to be EZ media.
Protein Gel Result
It looks like we can see the BSMT band, but the band does not have enough definition to say that J45181's BSMT is being expressed in greater quantity than J45120's BSMT.
Now, Alex says that it doesn't look like the GC will be available until next week.
Plate Reader Results
It looks like the machine screwed up. Barry is doing an injection test, and then, we will see what happens from there.
EZ Media Preparation
Made 500 mL 5X supplement EZ with the proper amount of amino acids in it plus .845 mL thiamine HCl minus calcium pantothenate, p-aminobenzoic acid, p-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid
Calculation for thiamine HCl
Recipe calls for .169 g in 25 mL or 6.76 mg/mL. Our stock is 10 mg/mL. Thus, we would add .676 the amount of our stock to the 100X VA Vitamin Solution, which is 25 mL of 500 mL, or a 1:20 dilution. .676*.05 = .0338. Then, we would add 50 mL of 100X VA Vitamin solution per 1000 mL 5X, so .0338 *50 mL/5 = .338 mL/200 mL 5X solution.
So, I did J45700, J45800, & MG1655 in EZ media, EZ media - vitamins, EZ media - vitamins - PHE - TYR -TRP in 10 mL cultures with .4 mL fructose added