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From 2010.igem.org

Contents 1 Andreas 1.1 Preparation of competent Top10 Escherichia coli cells 1.1.1 Materials 1.1.2 Procedures 2 Hassan

Andreas

Preparation of competent Top10 Escherichia coli cells

Continued from 21/6.
Based on the protocol of the Knight lab

Materials

CCMB80 buffer

• 10 mM KOAc pH 7.0
• 80 mM CaCl2.2H2O
• 20 mM MnCl2
• 10 mM MgCl2.6H2O
• 10% glycerol

Procedures

1. 250 ml LB inoculated with 2 ml from ON culture
   • Grown at 28°C until an OD(600) of ≈0.3
2. Cells spun down at 3000x g for 10 min in 4°C
3. Cells resuspended in 80 ml ice-cold CCMB80 buffer and kept on ice for 20 min:
4. Cells spun down with same settings
5. Cells resuspended in 10 ml new CCMB80 and kept on ice for 20 min
6. Competent cells aliquoted in 100ul aliquots and stored in -80°C.

Hassan

EuMelanin (EM)

1. Melanin Gene in recombinant E. Coli.
2. How IPTG effecting EM production.
3. Study this article: http://www.ncbi.nlm.nih.gov/pubmed/17040223 Lagunas-Muñoz VH et. al. 2006 :

   • Suggests that temperature and PH are the main factors effecting melanin production. they reached a 100% increase in melanin production by changing PH and Temp.
   • The production of melanin is subjected to the presence of tyrosinase, which is an enzyme to catalyze the oxidation of phenols (for the melanin case this one is tyrosine) source: wikipedia.
   • These enzymes have two activities: they use molecular oxygen to catalyse the hydroxylation of l-tyrosine to l-3,4- dihydroxyphenyl alanine (l-dopa) (cresolase activity); and then oxidizes this product to dopachrome (catecholase activity), which then non-enzymatically oxidizes and polymerizes to form melanin (Garcı´a-Borro´n and Solano 2002).
   • EuMel production is what they were interested in, EuMel is directly related to induction of MutMelA gene, which could be controlled by concentration of IPTG. They suggest a 0.1 mmol/litr IPTG for mel production.

   Ask wetlab team for what their PH, Temp, and IPTG is for their bacteria.

4. Possible pathway in EM production in E. Coli.
5. What else is needed to represent the whole as system!?

Overexpression / inclusion bodies

1. TODO:

   Read about inclusion bodies, over expression.

   Read articles in production part of modeling and add more/delete some.