IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/07/28: Difference between revisions
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# Mix all products and ligate | # Mix all products and ligate | ||
=== Restriction Digestion of I0500 and pSB3K3 === | |||
* Digest I0500 with SpeI | |||
* Digest pSB3K3 with EcoRI and PstI | |||
* Run on 0.8% agarose gel with SyBr Green along with uncut I0500 and pSB3K3 as control | |||
Result: | |||
* Failed to get any bands | |||
* Serial dilution in the preparation process and DNA loss in gel extraction resulted in low DNA concentration in well | |||
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Revision as of 08:08, 1 August 2008
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Plasmid AssemblyFur- MutantBrief Introduction of Fur Iron is an important nutrient for E.coli but it presents problems of toxicity at high concentration, poor solubility and low availability. Escherichia coli achieves iron homeostasis through regulation of numerous iron acquisition systems and iron storage proteins. This regulation is mediated by the Fur (ferric uptake regulation) protein, which generally acts as a repressor that utilises ferrous iron as a co-repressor. Fur also regulates genes involved in other functions such as acid shock response, defence against oxygen radicals, and virulence, and is therefore regarded as a global regulator. Thus, fur- mutants which have higher free iron ion concentration in the cytoplasm are to be used for the formation of magnetite in vivo. Preparing fur- Mutants Colonies
Preparing BiobricksGene (mms6 or mamC): File:Http://openwetware.org/images/9/98/Genecam.jpeg Promoter I0500 on plasmid pSB2K3: File:Http://partsregistry.org/wiki/images/thumb/a/a6/PSB2K3 map.jpg/800px-PSB2K3 map.jpg Plasmid pSB3K3: http://partsregistry.org/Part:pSB3K3 Plan:
Restriction Digestion of I0500 and pSB3K3
Result:
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