Team:Cambridge/Project/Melanin

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(Melanin Pigment)
 
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= Melanin Pigment =
= Melanin Pigment =
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[[#Background | Background]]
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[[#Design | Design]]
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[[#Characterisation | Characterisation]]
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== Introduction ==
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== Background ==
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'''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.
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.
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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]
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]
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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])
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'''MelA'''
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Currently our MelA gene is held on the following plasmid:
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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.
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[[Image:Mel plasmid mk 2.JPG]]
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=== Action plan of our team ===
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== Previous Work ==
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The plasmid we used was from Duncan's stocks. We isolated it using miniprep to make stocks of our own before transforming it into a biobrick.
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== Action plan of our team ==
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Our action plan is as follows:
Our action plan is as follows:
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:3. Integrate Mel biobrick into system (e.g amplification of logic gate system)
:3. Integrate Mel biobrick into system (e.g amplification of logic gate system)
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== Testing Activity of Melanin ==
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== Design==
 +
 
 +
 
 +
 
 +
===Constructing the BioBrick===
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'''Biobrick'''
 +
 
 +
Our aim is to make the MelA gene into a biobrick as follows:
 +
 
 +
[[Image:MelAbiobrick.jpg]]
 +
 
 +
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:
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 +
[[Image:MelA primer map.JPG]]
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 +
The PCR's will be done in the following steps, in order to remove both sites and prepare the gene as a biobrick:
 +
 
 +
[[Image:MelA.png]]
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 +
== Characterisation ==
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 +
'''Successful Pigment Production'''
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 +
In order to produce pigment, bacteria transformed with pTRCmelA must be plated on media supplemented with copper and tyrosine.  Supplementation with copper is necessary as copper is a cofactor for the tyrosinase, and tyrosine is a precursor to melanin biosynthesis.  (N. Cabrera-Valladares, A. Martínez, S. Piñero, V.H. Lagunas-Muñoz, R. Tinoco and R. de Anda et al., Expression of the melA gene from Rhizobium etli CFN42 in Escherichia coli and characterization of the encoded tyrosinase, Enzyme Microb Technol 38 (6) (2006), pp. 772–779 [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TG1-4H21NC4-4&_user=1495569&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=972838039&_rerunOrigin=scholar.google&_acct=C000053194&_version=1&_urlVersion=0&_userid=1495569&md5=3a4e939d82057cc796749e16cab13a3b])
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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. 
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Grew the plasmid in TOP10 E. coli
 
[[Image:SDC10545.JPG|500px]]
[[Image:SDC10545.JPG|500px]]
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Pigment was clearly produced.
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Left: Top10 transformed with plasmid containing the MelA gene.  Right: untransformed Top10.  
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== Constructing MelA biobrick ==
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'''Control Experiments'''
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'''Primer design''':
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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).
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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.
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'''Optimization'''
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'''PCR reactions'''
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On pTRCmelA, 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.
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Practical details to follow as soon as primers arrive.
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[[Image:Cambridge SDC12335.JPG | 300px]]
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== Characterisation of Biobricks and colour output ==
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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
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From left to right, bottom row: 0.075 mg/mL tyrosine, 0.3 mg/mL tyrosine, and then 0.6 mg/mL tyrosine.
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== Test compatibility with other biobricks ==
 
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Hopefully we will be able to integrate this into the amplification processing system.
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Latest revision as of 12:45, 21 October 2009


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.

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]

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:

MelAbiobrick.jpg

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:

MelA primer map.JPG

The PCR's will be done in the following steps, in order to remove both sites and prepare the gene as a biobrick:

MelA.png

Characterisation

Successful Pigment Production

In order to produce pigment, bacteria transformed with pTRCmelA must be plated on media supplemented with copper and tyrosine. Supplementation with copper is necessary as copper is a cofactor for the tyrosinase, and tyrosine is a precursor to melanin biosynthesis. (N. Cabrera-Valladares, A. Martínez, S. Piñero, V.H. Lagunas-Muñoz, R. Tinoco and R. de Anda et al., Expression of the melA gene from Rhizobium etli CFN42 in Escherichia coli and characterization of the encoded tyrosinase, Enzyme Microb Technol 38 (6) (2006), pp. 772–779 [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TG1-4H21NC4-4&_user=1495569&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=972838039&_rerunOrigin=scholar.google&_acct=C000053194&_version=1&_urlVersion=0&_userid=1495569&md5=3a4e939d82057cc796749e16cab13a3b])


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.

SDC10545.JPG

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

Control Experiments

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).

Optimization

On pTRCmelA, 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.

Cambridge SDC12335.JPG

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.


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