IGEM:MIT/2006/Notebook/2007-8-6

=Things to Do=

1. Add 100 uL fluorescent cultures to 25 mL media and grow them up at 220 RPM- DONE (8:00 AM)

2. Remove samples from fluorescent cultures in exponential phase and prepare them for protein gel while controlling for cell density (see below) and store the pellet at -80C in order to do prep in parallel with stationary phase samples- DONE


 * B0015 OD600=0.87, pelleted down 2.5/.87*2 mL = 5.75 mL culture


 * R0040.E0840 OD600=0.83, pelleted down 2.5/.83*2 mL = 6.02 mL culture (NOTE- had to be grown up on shaker 30 mins. longer than others while other sat in warm room)


 * J45995 OD600=1.17, pelleted down 2.5/1.17*2 mL=4.27 mL culture


 * J45996 OD600=1.16, pelleted down 2.5/1.16*2 mL=4.31 mL culture

3. Remove samples from fluorescent cultures in stationary phase and prepare them for protein gel- DONE


 * J45995 OD600=2.66, pelleted down 2.5/2.66*2 mL= 1.88 mL culture


 * J45996 OD600=2.86, pelleted down 2.5/2.86*2 mL= 1.75 mL culture


 * R0040.E0840 OD600=3.10, pelleted down 2.5/3.10*2 mL= 1.61 mL culture


 * B0015 OD600=3.10, pelleted down 2.5/3.10*2 mL = 1.61 mL culture

4. Smell J45250 + J45181 cultures in exponential and stationary phase- DONE (3K3 + 1AT3- mint in exponential phase with precursor smell (0.89 OD600 after 12 hours of growth), "poop with a hint of mint"- Jason in stationary phase or OD600=1.94); 1AC3 + 1AT3- mint in exponential phase with precursor smell with hint of banana (0.61 OD600 after 18 hours of growth), mint with a hint of banana maybe at OD600=1.64), will keep growing these

5. Autoclave waste- DONE

6. Make alternate glycerol stabs of indole-knockout J45120 and J45181 cultures- DONE

7. Make up EZ media -TRP -TYR -PHE without iron added- DONE

8. Grow up the following 100-mL cultures- DONE:


 * J45700 on EZ media -TRP -TYR -PHE -Fe + Fructose


 * J45800 on EZ media -TRP -TYR -PHE -Fe + Fructose


 * TOP10 on EZ media -TRP -TYR -PHE -Fe + Fructose


 * J45700 on EZ media + Glycerol


 * J45800 on EZ media + Glycerol


 * TOP10 on EZ media + Glycerol

9. Smell existent EZ media cultures that have grown up- DONE (smell like mint to me!)

10. Ask Reshma if I can stick cultures in fridge from exponential phase and prepare protein gel samples from stationary and exponential phase in parallel- DONE

11. Ask everyone if it is okay if I start trying to make a genetically modified leucine-overproducer- DONE

12. Miniprep and sequence J45250 3K3 and J45600 3C5- DONE (NOTE: could only do VF2 and VR because DNA yields low (low copy plasmids))

13. Make glycerols of variants of J45250 + J45181 and J45600 + J45800 along with J4520 3K3 and J45600 3C5- DONE (although let 250 + 181s grow up some more)

14. Obtain denaturing buffer from Reshma- DONE

15. Clean up lab area- DONE

16. Ask Dr. Esaki if ammonium formate can just be added to the media instead of in a reaction mix- DONE