Jared Maybee's blog

8-10-09
-Began LBB time-lapse assay with all mutants relative to wild type.
-Continuing paper and presentation

8-13-09

8-5-09
-Inoculated all mutant strains and WT into 2.5mL LB broth @10:10am @RT
-Finish final touches on Furse paper
-Continue Final paper
-Continue final presentation work

8-06-09
Gathered samples from a nearby river, two of them field-tested LBB positive.

7-27
-Inoculated all 7 mutant strains (E1, E2, E3, C1, C3, F1, D1) for 5 hours starting at 10:21am then plated onto YE% screening plates.
-Made LBKan (with proper amount of Kan - I used 6µl for the previous plates for some reason instead of 600µl) and YE% plates again. YE 225µl did not mix terribly well. It appeared to solidify too quickly.
-Repeat transformation (plated onto wrong amount of LBKan plates, so this will be redone tomorrow)
- O/N cultures for two transformants which did grow correctly (F1, D1).

7-13
Repeated conjugation procedure exactly as before except the strains are labeled H-P.

Repeated competent cell transformation with commercial competent cells (exactly the same as before).

7-14

7-6
The idea is to purify the plasmid that was inserted into competent Ecoli cells. We know they took up the plasmid because WT ecoli are not kan resistant, but all of the harvested bacteria was grown on LBKan plates. Competent Ecoli was plated to verify the plasmid was taken up.
Protocol for Plasmid DNA Purification:
1. Resuspend pelleted bacteria cells in 250µl Buffer P1 and transfer to a microcentrifuge tube
2. Add 250µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times

The following pictures represent a temperature screening for Mn oxidation for all seven of the IMO mutants along with a WT on each plate for comparison. WT did not oxidize whatsoever in 13C, whereas E2, C1, E1, and to some degree D1 and E3 all did oxidize.

The 27C plate displayed essentially full oxidation over the time period for all strains, thus not proving to be helpful over this timeframe.

There is no tab on my week 3 blog for view or edit, so I will continue it here instead.
The 'x' technique described in the previous blog was left for 24 hrs, the oxidation recorded, and then left for another 12 (36hrs total). The following pictures demonstrate the levels after this time period (the blue dots over the 'x' represent oxidatin after 24 hrs):

6-22
Of the mutants plated last week, B1, B2, B3, C1, C2, C3, C4, D1, D4, E1, E2, E3, F1, G3, H1, and H2 were determined to be IMO and thus now are going to be screened and characterized. The screening will begin as follows:

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