Week 4

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.

At Room Temperature, however, only WT, C3, and E3 managed to not become largely oxidized. It should be noted that these strains showed a similar lack of oxidation in the 13C plate, whereas the other strains all showed signs of oxidation.

The Procedure we used for making E.Coli (KG64 strain) competent is as follows:

1) Start the culture by incubating a single colony of the recipient strain into 5ml LB broth in a caped tube shaking @ 37°C

2) Subculture by inoculating 2.5ml of overnight culture into 250ml of LB broth. GRow this @37°C to an optical density of OD600=.3

3) Ice cells for 20 minutes, then transfer to two 200ml cold, sterilized centrifuge bottles

4) Centrifuge @2000g for 15 minutes @4°C

5) Remove supernatant and then resuspend int 50ml for each bottles (100ml total) of cold, sterile 100mM (.1M)CaCl2

6) Incubate for 20 minutes on ice

7) Centrifuge 2000g for 15 minutes @4°C

8) Resuspend in 2.5ml of cold, sterile CaCl2

9) Store in a single tube overnight (combine tubes before storing). In order to store, the technique we used was to submerge the bottles in ice inside an insulated container and place this container within the 4°C room. 

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10) Mix the chilled cell suspension and add 425µl of 80% sterile glycerol (final concentration 10%). Add glycerol slowly and over ice

11) Pipette .2ml aliquots into pre-chilled sterile microfuge tubes for storage at -70°C. Keep tubes on ice at all times.

Tranformation:

Using the ligated cells as described previously:
1) Thaw cells on ice
2) Add 5µl DNA and mix gently. Incubate for 30 mins on ice
3) Heat shock the cells for 2 min at 42°C and return to ice.
4) Add 1ml LB broth and incubate at 37°C for 1 hour.
5) Plate 200µl on selective media (LeptKan)