IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/07/28

Plasmid Assembly

Fur- Mutant

Brief 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

• fur- mutant strain received with Kanamycin resistance
• JW0669-2 CGSC~8758
• Filter paper with bacteria soaked with LB and plated on agar plates with and without Kanamycin (25μg/mL)

Preparing Biobricks

Gene (mms6 or mamC):

Plasmid map for pSB2K3

Plasmid pSB3K3: http://partsregistry.org/Part:pSB3K3

Plan:

1. Cut promoter I0500 with SpeI
2. Gel electrophoresis to purify promoter DNA
3. Cut mms6/mamC gene with XbaI and ligate to the promoter as the SpeI and XbaI sites bind
4. Cut the promoter and gene with EcoRI
5. Cut the plasmid with EcoRI and PstI
6. Run gel for products 4 and 5 to check size of bands
7. 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
• Control lanes have been diluted too (5μLDNA + 11μLSDW + 4μL dye)