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Team:Cambridge/Project/Melanin
From 2009.igem.org
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
Background
Melanin Production
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]
MelA
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 creates a Proline to Serine mutation that 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]) The plasmid was provided by Christine Sanntos from the lab of G. Stephanopoulos to Duncan Rowe under the materials transfers agreement.
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)
Design
Constructing the BioBrick
Biobrick
Our aim is to make the MelA gene into a biobrick as follows:
In order to do so, we had to remove forbidden restriction sites within the gene using primers and the in-fusion PCR technique. Primers were ordered as shown by the image below, to add the prefix and suffix and change the PstI restriction site from CTGCAG to CTGCAA:
The PCR's will be done in the following steps, in order to remove both sites and prepare the gene as a biobrick:
