Team:Cambridge/Project/Melanin

From 2009.igem.org

(Difference between revisions)
(Testing Activity of Melanin)
(Constructing MelA biobrick)
Line 84: Line 84:
Pigment was clearly produced.
Pigment was clearly produced.
-
== Constructing MelA biobrick ==
+
== 2. Constructing MelA biobrick ==
'''Primer design''':
'''Primer design''':

Revision as of 13:13, 4 August 2009


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.

Tyrosinase action.jpg

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.

Mel plasmid mk 2.JPG

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 SDC10545.JPG

Pigment was clearly produced.

2. 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.

Characterisation of Biobricks and colour output

Test compatibility with other biobricks

Hopefully we will be able to integrate this into the amplification processing system.