Getting Promoter Plasmid and Iron Assay with Fur- Mutants
Promoter Plasmid
- No colonies formed on the Kanamycin plates for I0500 and pUC control still
- Re-selected promoter plasmid R0010 with ampicillin resistance in well 5E on plate 1002
- Extracted R0010 from filter paper following standard protocol
- Transform competent TOP10 with R0010 and J63006 again following standard transformation protocol with some amendments
- R0010: 5μL DNA + 25μL TOP10 + 55μL SOC
- J63006: 15μL DNA + 25μL TOP10 + 45μL SOC
- Plate out neat and 1/10 dilution on freshly prepared ampicillin plates of 100μg/mL
- Incubate overnight at 37°C
Iron Assay with Fur- Mutants
- Added 0.222g of iron sulphate into 400mM sodium citrate stock prepared beforehand
- Notice that some iron sulphate solid cannot dissolve into solution completely which may affect iron concentration slightly
- Plastic ware used throughout to prevent alteration of iron concentration
- pH paper used to test iron stock to be around pH7
Protocol for Varying Iron Concentration in Medium
[Fe]/μM |
0 |
4 |
8 |
12 |
16 |
20
|
Vol. of Fe stock/ml
|
0 |
0.01 |
0.02 |
0.03 |
0.04 |
0.05
|
Vol. of SDW/ml
|
0.50 |
0.49 |
0.48 |
0.47 |
0.46 |
0.45
|
Vol. of fur- cells in M9/ml
|
9.5 |
9.5 |
9.5 |
9.5 |
9.5 |
9.5
|
- Placed plastic tubes with cells and medium inside the plastic jar borrowed from the Path Dept
- Add SDW into package containing chemical reagents and catalyst to activate it
- Package produces hydrogen gas which reacts with oxygen in air in the presence of palladium catalyst
- Carbon dioxide also produced to form a CO2-enriched environment, favouring anaerobic growth by E.coli
- Cultures incubated at 37°C for 2 nights
|