MG1655 grew on all the different M9 minimal media mixtures (casamino acids + fructose, casamino acids + glycerol, casamino acids + glucose, fructose, glycerol, & glucose), although on glycerol with no casamino acids, the culture only grew to 0.03 overnight
Things to Do Today
1. Take out R0040.E0840, J45995, J45996, & B0015 (11:15; 17:15 of growth) and add 100 uL culture to 25 mL media in 250 mL baffled flasks (started growing at 11:30)- DONE
2. Take OD600 measurements of R0040.E0840, J45995, J45996, & B0015 at 3:30 (became 3:50)- SEE BELOW
3. Run protein gel of samples collected yesterday- DONE
4. Grow up J45800 and J45700 on amino acid-supplemented EZ media and MG1655 (no antibiotics) (~4:15)- DONE
5. Retry growing J45800 and J45700 on casamino acid-supplemented fructose M9 media (~4:50) and leucine, proline, alanine, aspartic acid, serine-supplemented fructose M9 media- DONE (~7:45)
Recipe: http://openwetware.org/wiki/Endy:M9_media/supplemented, except 40 mL fructose (to be added individually to culture) + 389.8 mL H20
For LPAAS-suplemented fructose M9 media, I filter sterilized 100 mL of the media-casamino acids with the following additions:
- .142 g proline
- .114 g aspartic acid
- .0392 g alanine
- .0655 g serine
- .1221 g leucine
- ALL calculations from defined media Tom found except for leucine.
- The amount of leucine to be added calculated from casamino acid recipe http://www.usbio.net/Product.aspx?ProdSku=C2080
Recipe calls for 9.9% proline, 6.3% aspartic acid, 2.8% alanine, 5.6% serine, and 8.4% leucine...
So, I found the amount of leucine to add depending on each ratio between amino acid x and leucine...
Comparing with proline .142 g * 8.4/9.9 = .12048, aspartic acid .114 g* 8.4/6.3 = .152 g, alanine .0392 g* 8.4/2.8 = .1176, serine .0655 g * 8.4/5.6 = .09825.
Then, I took the average of these four figures to yield that I should add .1221 g leucine.
6. Meeting on BE grad school (10 AM)- DONE
OD600s after 4:20 growth
Based on this,
Here are the dilutions into 125-mL flasks:
1 mL J45995, 24 mL media
1 mL J45996, 24 mL media
1.45 mL B0015, 23.55 mL media
1.93 mL R0040.E0840, 23.07 mL media
Grow up for 45 mins
Then put in plate reader
- NOTE- due to a small delay in action of the plate reader, the run was delayed ~90 mins