Team:Cambridge/Project/Pigments

From 2009.igem.org

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(→The Kit of Parts)
(→The Kit of Parts)
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As as a result of our efforts, this summer, we have submitted the following biobricks to the Registry.  We hope that in the future this this kit of parts will be expanded in the future.
As as a result of our efforts, this summer, we have submitted the following biobricks to the Registry.  We hope that in the future this this kit of parts will be expanded in the future.
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<!--DONT EDIT THIS BIT:---------------------------------------------------------------------------->
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{| style="color:#CCC; background-color:#3D5089;" cellpadding="6" cellspacing="0" border="1"
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! Registry Code
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! Type
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! Sequence Description / Notes
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! Length
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|- style="color:#333; background-color:#A3C3FF;" cellpadding="6" cellspacing="0" border="1"
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<!----------------------------------------EDIT HERE ONWARDS---------------------------------------->
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| <partinfo>BBa_K274001</partinfo>
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| Reporter
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| '''MelA'''. The gene (melA) codes for a tyrosinase which produces a dark brown pigment from L-tyrosine. Production of the pigment requires the addition of copper and L-tyrosine supplements (the copper acts as a cofactor for the gene product) but no other precursors. The BioBrick sequence includes the native ribosome binding site.
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| 1844bp
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|- style="color:#333; background-color:#A3C3FF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K274002</partinfo>
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|Reporter
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|'''Violacein'''. Produces a purple pigment (violacein) from L-tyrosine. The operon contains five genes (VioA-E) each with their own ribosome binding sites.
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| 7346bp
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|- style="color:#333; background-color:#A3C3FF;" cellpadding="6" cellspacing="0" border="1"
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|<partinfo>BBa_K274003</partinfo>
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|Reporter
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|'''Vio operon ABDE'''. Produces a dark green pigment from L-tyrosine. Formed from the vio operon biobrick (BBa_K274002) with the vioC gene removed by restriction digest with BamHI and BglII. This sequence contains four genes, vioA, vioB vioD and vioE, each preceded by their own ribosome binding site.
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| 6032bp
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|- style="color:#333; background-color:#A3C3FF;" cellpadding="6" cellspacing="0" border="1"
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|<partinfo>BBa_K274004</partinfo>
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|Reporter
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|'''Vio operon ABCE'''. Produces a light green pigment from L-tyrosine. Formed from the vio operon biobrick (BBa_K274002) with the vioD gene removed by restriction digest with BglII and BclI. This sequence contains four genes, vioA, vioB vioC and vioE, each preceded by their own ribosome binding site.
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| 6200bp
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|- style="color:#333; background-color:#A3C3FF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K274100</partinfo>
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| Composite
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| '''CrtEBI with rbs'''. This Composite Biobrick is created by standard assembly of 3 basic Biobricks coding for enzymes CrtE, CrtB and CrtI (each with rbs). Together, enzymes CrtE, CrtB and CrtI convert colourless farnesyl pyrophosphate to '''red lycopene''' (via intermediates geranylgeranyl pyroiphosphate and phytoene). The red lycopene pigment can then be used as a coloured signal output, e.g. for biosensors.
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| 3385bp
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|- style="color:#333; background-color:#A3C3FF;" cellpadding="6" cellspacing="0" border="1"|
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| <partinfo>BBa_K274110</partinfo>
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| Generator
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| '''CrtEBI under constitutive promoter'''. This Biobrick is created by putting enzyme cassette CrtEBI (with individual rbs) of Part BBa_K274100 under constitutive promoter R0011. Amount of lycopene produced can be measured by photospectrometer with absorbance at 475nm (lycopene extraction using acetone).
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| 3448bp
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|- style="color:#333; background-color:#A3C3FF;" cellpadding="6" cellspacing="0" border="1"|
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| <partinfo>BBa_K274120</partinfo>
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| Generator
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| '''CrtEBI under pBad promoter'''. This Biobrick is created by putting enzyme cassette CrtEBI (with individual rbs) of Part BBa_K274100 under arabinose-induced promoter I0500. Amount of lycopene produced can be measured by photospectrometer with absorbance at 475nm (lycopene extraction using acetone). 
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| 4603bp
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|- style="color:#333; background-color:#A3C3FF;" cellpadding="6" cellspacing="0" border="1"
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| <partinfo>BBa_K274200</partinfo>
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| Composite
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| '''CrtEBIY with rbs'''. This Composite Biobrick is created by standard assembly of 4 basic Biobricks coding for enzymes CrtE, CrtB, CrtI and CrtY (each with rbs). Together, enzymes CrtE, CrtB, CrtI and CrtY convert colourless farnesyl pyrophosphate to '''orange beta-carotene''' (via intermediates geranylgeranyl pyroiphosphate, phytoene and lycopene). The orange beta-carotene pigment can then be used as a coloured signal output, e.g. for biosensors.
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| 4555bp
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|- style="color:#333; background-color:#A3C3FF;" cellpadding="6" cellspacing="0" border="1"|
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| <partinfo>BBa_K274210</partinfo>
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| Generator
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| '''CrtEBIY under constitutive promoter'''. This Biobrick is created by putting enzyme cassette CrtEBIY (with individual rbs) of Part BBa_K274200 under constitutive promoter R0011. Amount of beta-carotene produced can be measured by photospectrometer with absorbance at 455nm (beta-carotene extraction using acetone).
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| 4618bp
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|- style="color:#333; background-color:#A3C3FF;" cellpadding="6" cellspacing="0" border="1"|
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| <partinfo>BBa_K274220</partinfo>
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| Generator
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| '''CrtEBIY under pBad promoter'''. This Biobrick is created by putting enzyme cassette CrtEBIY (with individual rbs) of Part BBa_K274200 under arabinose-induced promoter I0500. Amount of beta-carotene produced can be measured by photospectrometer with absorbance at 455nm (beta-carotene extraction using acetone).
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| 5773bp
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|- style="color:#333; background-color:#A3C3FF;" cellpadding="6" cellspacing="0" border="1"|
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| <partinfo>BBa_K274111</partinfo>
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| Composite
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| '''CrtY under Pbad promoter (I0500)'''. This construct is created by putting BBa_K118013 (rbs+CrtY) under the control of Pbad promoter (I0500), by standard assembly. When used in double-transformation together with a constitutive lycopene-producing device (e.g. BBa_K274110), the resulting system can produce colour change (red to orange) upon induction with arabinose.
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| 2380bp
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|- style="color:#333; background-color:#A3C3FF;" cellpadding="6" cellspacing="0" border="1"|
==Choosing Pigments==
==Choosing Pigments==

Revision as of 17:52, 21 October 2009


Pigments

The Kit of Parts

Though E. coli does not naturally produce pigment, several other bacterial species secrete pigmented antibiotics. We mined bacterial genomes for pigment-producing operons, and transformed the most prominent candidates into E. coli. In particular, we have devoted our summer to 3 different pigment systems:

  • Carotenoids: The enzymes required for carotenoid production originally come from Pantoea ananatis, and were available in the registry. We used them to produce orange and red.
  • Melanin: The tyrosinase required for melanin production originally comes from Rhizobium etli and produces brown.
  • Violacein: The enzymes required for voilacein production originally come from Chromobacterium violacein. The operon can be manipulated to produce voilet, green, and blue.

As as a result of our efforts, this summer, we have submitted the following biobricks to the Registry. We hope that in the future this this kit of parts will be expanded in the future.

Choosing Pigments

We chose to pursue these 3 pigment systems for two main reasons.

Visual Diversity: Not only do these devices come from different bacterial species, but between these systems we've almost made -- as cheesey as it sounds -- all the colours of the rainbow.

A COOL PICTURE HERE

Design: We were able to use a variety of different techniques to build and design these pigment systems as biobricks. The carotenoid parts were already in the Registry, and were used standard assembly protocols to build pigment producing devices. The melanin system is a single gene which we endeavoured to turn into a biobrick by PCR. Finally, we designed the violacein operon for synthesis, codon optimizing it for both E. coli and B. subtilis and designing it so it is easy to manipulate to generate different pigments.

Potential for Gene Manipulation: The melanin system is particularly elegant in that a single gene is able to generate pigment as long as the bacteria are supplemented with copper and tyrosine. On the flip side, the carotenoid and violacein systems are multi-gene operons that make more than one pigment each. Further, they generate pigments without any supplements to the media.

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Registry Code Type Sequence Description / Notes Length
Reporter MelA. The gene (melA) codes for a tyrosinase which produces a dark brown pigment from L-tyrosine. Production of the pigment requires the addition of copper and L-tyrosine supplements (the copper acts as a cofactor for the gene product) but no other precursors. The BioBrick sequence includes the native ribosome binding site. 1844bp
Reporter Violacein. Produces a purple pigment (violacein) from L-tyrosine. The operon contains five genes (VioA-E) each with their own ribosome binding sites. 7346bp
Reporter Vio operon ABDE. Produces a dark green pigment from L-tyrosine. Formed from the vio operon biobrick (BBa_K274002) with the vioC gene removed by restriction digest with BamHI and BglII. This sequence contains four genes, vioA, vioB vioD and vioE, each preceded by their own ribosome binding site. 6032bp
Reporter Vio operon ABCE. Produces a light green pigment from L-tyrosine. Formed from the vio operon biobrick (BBa_K274002) with the vioD gene removed by restriction digest with BglII and BclI. This sequence contains four genes, vioA, vioB vioC and vioE, each preceded by their own ribosome binding site. 6200bp
Composite CrtEBI with rbs. This Composite Biobrick is created by standard assembly of 3 basic Biobricks coding for enzymes CrtE, CrtB and CrtI (each with rbs). Together, enzymes CrtE, CrtB and CrtI convert colourless farnesyl pyrophosphate to red lycopene (via intermediates geranylgeranyl pyroiphosphate and phytoene). The red lycopene pigment can then be used as a coloured signal output, e.g. for biosensors. 3385bp
Generator CrtEBI under constitutive promoter. This Biobrick is created by putting enzyme cassette CrtEBI (with individual rbs) of Part BBa_K274100 under constitutive promoter R0011. Amount of lycopene produced can be measured by photospectrometer with absorbance at 475nm (lycopene extraction using acetone). 3448bp
Generator CrtEBI under pBad promoter. This Biobrick is created by putting enzyme cassette CrtEBI (with individual rbs) of Part BBa_K274100 under arabinose-induced promoter I0500. Amount of lycopene produced can be measured by photospectrometer with absorbance at 475nm (lycopene extraction using acetone). 4603bp
Composite CrtEBIY with rbs. This Composite Biobrick is created by standard assembly of 4 basic Biobricks coding for enzymes CrtE, CrtB, CrtI and CrtY (each with rbs). Together, enzymes CrtE, CrtB, CrtI and CrtY convert colourless farnesyl pyrophosphate to orange beta-carotene (via intermediates geranylgeranyl pyroiphosphate, phytoene and lycopene). The orange beta-carotene pigment can then be used as a coloured signal output, e.g. for biosensors. 4555bp
Generator CrtEBIY under constitutive promoter. This Biobrick is created by putting enzyme cassette CrtEBIY (with individual rbs) of Part BBa_K274200 under constitutive promoter R0011. Amount of beta-carotene produced can be measured by photospectrometer with absorbance at 455nm (beta-carotene extraction using acetone). 4618bp
Generator CrtEBIY under pBad promoter. This Biobrick is created by putting enzyme cassette CrtEBIY (with individual rbs) of Part BBa_K274200 under arabinose-induced promoter I0500. Amount of beta-carotene produced can be measured by photospectrometer with absorbance at 455nm (beta-carotene extraction using acetone). 5773bp
Composite CrtY under Pbad promoter (I0500). This construct is created by putting BBa_K118013 (rbs+CrtY) under the control of Pbad promoter (I0500), by standard assembly. When used in double-transformation together with a constitutive lycopene-producing device (e.g. BBa_K274110), the resulting system can produce colour change (red to orange) upon induction with arabinose. 2380bp
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