IGEM:MIT/2006/Notebook/2007-8-2

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Contents

OD600s for J45700, J45800, and MG1655

EZ + Glycerol

After 17 hours

J45700- 0.02

J45800- 0.00

MG1655- >2.00

After 22 hours

J45700- 0.74

J45800- 0.34

M9 Minimal + Casamino Acids + Fructose

After 18 hours

J45700- 0.00

J45800- 0.00

MG1655- >2.00

After 23 hours

J45700- 0.03

J45800- 0.00

M9 Minimal + Alanine, Aspartic Acid, Leucine, Proline, Serine + Fructose

After 14 hours

J45700- 0.00

J45800- 0.01

MG1655- 0.01

After 19 hours

J45700- 0.00

J45800- 0.00

MG1655- 0.03

  • 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.

Image:070802gel1.tif

Image:070802gel2.tif


Banana Update

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

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