IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/08/06

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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

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