Team:UoA NewZealand/Experiments
11/8 First Result ever: Found that the DE3 E. coli cells we were using were intrinsically resistant to chloramphenicol. Couldn’t tell using the iGEM competency test (fancy name for it?) (which added a plasmid with chloramphenicol resistance) how competent our cells were, as those with the plasmid included would have been selected against. Switched to DH5 alpha cells for further recombination.
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- Were they chemically competent?
- They were, calculations to the competency on page 12 of lab book 06/09
- Also other competent cells - 09/09
- gels/photos would be gr9
- Optional: mention the fricking RE sites in the middle of the construct?
- 16/08 (labbook) - Optional: talk about issues with ligation/understanding provided igem protocol
- Gel - 31/08
- 03/09 - 04/09 - made new chemically competent cells
- Gained PCR skills 07/09, 08/09
Cells passed competency test, but really struggled to take up our construct, switched to electroporation as recommended by our PI, and as it would remove more steps that could go wrong in chemical transformation.
23/09 Kan backbone + PETase End colonies grew on plates 1) Kan plate and competent cells - no growth 2) + 3) - PETase end + kanamycin backbone - many small colonies 4) negative control - kan plate and no cells - no growth 5) plain agar plate with cells ‘transformed’ with PETase end (to see growth of cells after transformation) - lawn of cells
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Miniprep of kan backbone + PETase end -> 27/09, gel page 20, used another miniprep protocol 29/09 - still no DNA
- Could be due to overgrowing colonies
- Unlikely, cells displayed kanamycin resistance when innoculated in higher concentration (80ng/ul vs 50ng/ul) and when grown on fresh plates
- Grew cells on someone else’s kanamycin - still grew
- Could be due to fault in construct - EcoRI site that would have cut up the PETase end
- Self-ligation of the backbone?
- Backbone insertion into the DNA?
- WHY
Decided to use fresh DH5 alpha cells and to ditch the faulty construct
03/10 Third Result: electroporation results Decided to use another backbone (pSB1C3) as gel (page 34 - 04/10) showed that PCR of kan backbone not entirely pure and was worried with the previous results of the plasmid being able to insert into the genome/being low copy number/self-ligating. Successfully transformed cells with plasmid given by one of our advisors & confirmed with miniprep page 44 - therefore transformation is not the problem 1 = ampicilin plate with cells transformed with peroxyredoxin plasmid- many big colonies 2 = kan backbone only transformants - no growth 3 = col backbone with PETase start -no growth Did some transformations with cells provided by our PI - as they were known to be electrocompetent. Also used a plasmid with a multiple cloning site we could use for our construct, pET-DUET to see whether that would take up our construct any easier. 4 = chloramphenicol plate with col backbone digest at 9/10 dilution (no insert) - lawn of plates - issue with antibiotic plating/spreading 5 = ampicilin plate + petduet + PETase - no growth 6 = canvas 7 =
3 = chlorampheicol plate with col backbone + PETase start insert -
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Forth Results: start monitoring on the digestion and ligation concentration - put on gel to see results.
1) 2)3)4)5)6)7)8)9)10)11)12)13)14) => 2 positive controls are positive, one colony growth in negative control and two colonies in insert + backbone (PETDUET)
