TO DO: *Electroporate Dr. Fray's plasmid containing aiiA into DH5a If any colonies grow until evening - culture up for testing on Friday !! *Maxi & mini of J37022, aiiA (with flag tag), aiiA *Next ligation step for new predator cell (with the new aiiA) once maxiprep is finished & electroporation on same day ! *Testing of J37015, J37022, Acylace Activity (need J37016 for two of them) *Send J37022 for sequencing *Make frozen stock of new T9002, J37036, J37022 !!! *Control digest for J37036 - Has ligation worked?
*JS: Also, you guys need to start sending in parts to the registry, or else we won't have anything to show for all our efforts this summer!
*JW: Sorted out the fridge today - we have all the maxis, ready to be sent when necessary, however we have multiple copies of some. We need to determine which is the one that was used in construction.
- The new aiiA was sent to us in DH5-α. However the two promoters in the pGEM cloning vector in which it was sent (T7 and Sp6) do not work in DH5-α cells, and so attempting to test for expression here would be pointless
- We can however test for expression in Rosetta, which can use the T7 promoter.
Miniprep & Maxiprep
- Are the results successful?
- All four J37022 cultures successfully maxipreped
- One of the two Aiia cultures successfully maxipreped. DNA used for ligations
Ligation & electroporation
- New J37023 (Flagged and tagged Aiia from Dr Fray) insert ligated into previously maxipreped stock of 1I (B0015).
- Part J37024
- Electroporated at 5.15pm.
Electroporation of Dr. Fray's plasmid containing aiiA
- Was electroporated this morning into Rosetta (not into DH5a because no expression will take place in DH5a due to the promoter not working in this strain)
- Might get some colonies so that it can be cultured for Western Blot tomorrow
- Testing was carried out partly according to the current J37015 protocol and partly with some added measurements and amended steps. These are outlined below. (The protocol will be adjusted accordingly)
- Put into 2hrs shake at 9:45
- Taken out of shaker at 12:00
- Measure OD: 1.099 and dilute into 25mL to OD 0.1
- Spin down the cells & discard supernatant
- Add 25mL of prewarmed LB & resuspend
- Made up 2 x 25mL cultures
- One is inoculated with 1nM AHL (add 5μL of 5uM stock to the 25 mL)
- The other one is not inoculated with AHL
In Dr. Mann's Lab:
- Place in shaker
- Two samples were taken every 10-15min. for 2hrs
- One sample: Used Wallac-Victor III after taking samples to read fluorescence and OD (in order to detect GFP levels)
- Second sample: Supernatant is stored in order to assess AHL levels with J37016 later
Assessing AHL of samples with J37016:
- Replaced supernatant of J37016 with saved supernatant from above
- Put into shaker for 4hrs shake at 16:00
- Taken out of shaker at 19:30
- Fluorescence reading to assess amounts of AHL present (using J37016) afterwards
This was done to check whether acylase degrades anything that is vital to DH5a
- Put 2 x 2mL J37016 (OD 0.1) in shaker at 16:20
- One of them is inoculated with 8μL of Soln 1 of the acylase
- No enzyme was added to the other culture (control)
- OD will be checked & compared after a few hours in shaker
Control digest of J37036
- Identified enzymes - will be run first thing tomorrow morning
- Smaller fragment worked.....at last and so we will give the PCR fusion a go tomorrow