Team:Cambridge/Project/VI02

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= Violacein Pigments =
= Violacein Pigments =
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[https://2009.igem.org/Team:Cambridge/Project/VI01 Background]
[https://2009.igem.org/Team:Cambridge/Project/VI01 Background]
[https://2009.igem.org/Team:Cambridge/Project/VI02 Design]
[https://2009.igem.org/Team:Cambridge/Project/VI02 Design]
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== Design ==
== Design ==
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The Vio operon had numerous forbidden restriction sites.  We thus had to synthesize it, removing these restriction sites and optimizing codon usage for E. coli, to create the following biobrick:
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The Vio operon had numerous forbidden restriction sites, far too many to remove by PCR.  We thus decided to synthesize it. This allowes us too remove these restriction sites and optimize codon usage for E. coli, to create the following biobrick:
[[Image:violaceinoperon.jpg]]
[[Image:violaceinoperon.jpg]]
-
As DNA2.0 very generously agreed to synthesise the entire operon for us, we designed it to include all the five genes, each preceded by a ribosome binding site, and flanked by the prefix and suffix. All forbidden restriction sites were removed from the operon. The final plan for the inserted operon is shown below:
+
As DNA2.0 very generously agreed to synthesize the entire operon for us, we designed it to include all the five genes, each preceded by a ribosome binding site, and flanked by the prefix and suffix. The final plan for the inserted operon is shown below:
[[Image:Design sent to DNA 2.0.PNG]]
[[Image:Design sent to DNA 2.0.PNG]]
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This will be held under a repressible promoter on the pJexpress cloning cassette from DNA2.0. The restriction sites will allow us to easily remove the vioC and vioD which are the genes responsible for chaging pigment colour.
+
This will be held under a repressible promoter on the pJexpress cloning cassette from DNA2.0. We codon optimised the operon for both ''E. coli'' and ''B. subtilis'', and designed it to include restriction sites with complementary sticky ends around vioD and vioC. This allowed us to remove both genes easily to create more colours from the vio operon.
===Creating colours===
===Creating colours===
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Once the violacein biobrick arrived we expressed in in TOP10 E. coli to create the purple pigment. We then carried out two more digests to see if we could create further colours:
+
Once the violacein biobrick arrived we expressed in in TOP10 ''E. coli'' to create the purple pigment. We then carried out two more digests to see if we could create further colours:
:*BamHI and BglII = removed vioC and produced a dark green pigment
:*BamHI and BglII = removed vioC and produced a dark green pigment
:*BglII and BclI = removed vioD and produced a light green pigment
:*BglII and BclI = removed vioD and produced a light green pigment
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 +
With more time, we would have been able to use this system to create colour logic gates; where different inputs would create a mix of different coloured outputs. This is discussed further on the [https://2009.igem.org/Team:Cambridge/Future | Future] page, where we explore the different potentials and implications of our project.
 +
Both of these new pigments were entered into the registry, along with the whole violacein operon for the purple pigment.
Both of these new pigments were entered into the registry, along with the whole violacein operon for the purple pigment.
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===Biobrick Parts===
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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_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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|}
{{Template:CambridgeBottom}}
{{Template:CambridgeBottom}}

Latest revision as of 01:19, 22 October 2009


Violacein Pigments


Design

The Vio operon had numerous forbidden restriction sites, far too many to remove by PCR. We thus decided to synthesize it. This allowes us too remove these restriction sites and optimize codon usage for E. coli, to create the following biobrick:

Violaceinoperon.jpg

As DNA2.0 very generously agreed to synthesize the entire operon for us, we designed it to include all the five genes, each preceded by a ribosome binding site, and flanked by the prefix and suffix. The final plan for the inserted operon is shown below:

Design sent to DNA 2.0.PNG

This will be held under a repressible promoter on the pJexpress cloning cassette from DNA2.0. We codon optimised the operon for both E. coli and B. subtilis, and designed it to include restriction sites with complementary sticky ends around vioD and vioC. This allowed us to remove both genes easily to create more colours from the vio operon.

Creating colours

Once the violacein biobrick arrived we expressed in in TOP10 E. coli to create the purple pigment. We then carried out two more digests to see if we could create further colours:

  • BamHI and BglII = removed vioC and produced a dark green pigment
  • BglII and BclI = removed vioD and produced a light green pigment

With more time, we would have been able to use this system to create colour logic gates; where different inputs would create a mix of different coloured outputs. This is discussed further on the | Future page, where we explore the different potentials and implications of our project.

Both of these new pigments were entered into the registry, along with the whole violacein operon for the purple pigment.

Biobrick Parts

Registry Code Type Sequence Description / Notes Length
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


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