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Team:Cambridge/Project/Melanin
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Grew the plasmid in TOP10 E. coli | Grew the plasmid in TOP10 E. coli | ||
Revision as of 13:12, 4 August 2009
Categories :
Project :
-
Overview
Sensitivity Tuner
--- Characterisation
--- Modelling
Colour Generators
--- Carotenoids (Orange/Red)
--- Melanin (Brown)
--- Violacein (Purple/Green)
The Future
Safety
Notebook :
Team Logistics :
Melanin Pigment
Introduction
The MelA gene codes for a tyrosinase. Tyrosinases catalyze two reactions, as described in the figure below. Melanin is a macromolecular compound produced by the polymerization of the quinone product of the second reaction, and has a characteristic brown colour.
From H. Claus and H. Decker, Bacterial tyrosinases, Syst. Appl. Microbiol. 29 (2006), pp. 3–14. [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B7GVX-4H21K91-1&_user=1495569&_coverDate=01%2F24%2F2006&_fmt=full&_orig=search&_cdi=20442&view=c&_acct=C000053194&_version=1&_urlVersion=0&_userid=1495569&md5=de451c5e1d1d18ad12b7e39b10b80408&ref=full]
Our MelA gene is from Rhizobium etli. Further, it is a mutant; it has a C to T substitution at the 1,000th nucleotide, which reduces the amount of time before melanin production is visible. (Santos, C. N., and G. Stephanopoulos. 2008. Melanin-based high-throughput screen for L-tyrosine production in Escherichia coli. Appl. Environ. Microbiol. 74:1190-1197 [http://aem.asm.org/cgi/reprint/74/4/1190])
Previous Work
Currently our MelA gene is held on the following plasmid, provided by Duncan Rowe.
We isolated it using miniprep to make stocks of our own.
Action plan of our team
Our action plan is as follows:
- 1. Test for melanin production
- 2. Isolate MelA gene in biobrick form
- 3. Integrate Mel biobrick into system (e.g amplification of logic gate system)
1. Testing Activity of Melanin
Grew the plasmid in TOP10 E. coli
Pigment was clearly produced.
Constructing MelA biobrick
Primer design:
Primers were designed to isolate the melanin with the prefix and suffix. We are currently looking at adding a ribosome binding site to the the primers in order to produce a complete biobrick.
PCR reactions
Practical details to follow as soon as primers arrive.
