Revision as of 13:54, 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.

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, pTRCmelA, 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. Melanin Production

Proof of pigment production

We transformed Top10 E. coli with pTRCmelA and plated them, using LA supplemented with 15ug/ml copper, 0.2ug/ul tyrosine. Below is a plate that was incubated at 37 degrees for 24 hours and then left on the bench at room temperature over the weekend. Pigment was clearly produced, and it appears to have diffused out of the colonies.

Left: Top10 transformed with plasmid containing the MelA gene. Right: untransformed Top10.

To show that the brown colour was a result of the MelA gene and not natural oxidation of the LA supplements, a control plate without any bacteria was incubated for the same amount of time and showed no change in colour (data not shown).

On Duncan's plasmid, the MelA gene is under the control of the lac repressor. The photo above shows leaky expression of the promoter, as no IPTG was not added. We then experimented with the addition of IPTG and varying tyrosine concentrations to see the effect on pigment production. As the photo below shows, the greatest pigment production was achieved with IPTG induction and the highest concentration of tyrosine.

From left to right, top row: 1mM IPTG with 0.075 mg/mL tyrosine, 1mM IPTG and 0.3 mg/mL tyrosine, and then 1mM IPTG and 0.6 mg/mL tyrosine From left to right, bottom row: 0.075 mg/mL tyrosine, 0.3 mg/mL tyrosine, and then 0.6 mg/mL tyrosine.

Pigment Characterization

2. Constructing MelA biobrick

Biobrick

Primer design:

Primers were designed to isolate the melanin with the prefix and suffix. We kept the MelA gene's native ribosome binding site.

PCR reactions

Practical details to follow as soon as primers arrive.

3. Test compatibility with other biobricks

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

@@ Line 62: / Line 62: @@
 == 2. Constructing MelA biobrick ==
+'''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.
+Primers were designed to isolate the melanin with the prefix and suffix. We kept the MelA gene's native ribosome binding site.
 '''PCR reactions'''
 Practical details to follow as soon as primers arrive.
 == 3. Test compatibility with other biobricks ==
 Hopefully we will be able to integrate this into the amplification processing system.

Team:Cambridge/Project/Melanin

From 2009.igem.org