Team:Cambridge/Bioluminescence/Bacterial Codon optimisation

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{{:Team:Cambridge/Templates/headerMinimalprototype}}
{{:Team:Cambridge/Templates/headerMinimalprototype}}
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{{:Team:Cambridge/Templates/headerbar|colour=#386abc|title=Bacterial Codon Optimisation}}
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{{:Team:Cambridge/Templates/headerbar|colour=#386abc|title=Project Vibrio: Bacterial Codon Optimisation}}
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=What is codon usage?=
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{{:Team:Cambridge/Templates/Topheader|header=What is codon usage?}}
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[[Image:codons.jpg|300px|right|the universal genetic code]]
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{{:Team:Cambridge/Templates/rightpic|src=codons.jpg|caption=The universal genetic code}}
One of the fascinating features of life is the '''universal genetic code'''.  In all known organisms, from bacteria to man, the same triplets of DNA bases code for the same amino acids.  However this does not mean that all species encode their genomes in exactly the same way.   
One of the fascinating features of life is the '''universal genetic code'''.  In all known organisms, from bacteria to man, the same triplets of DNA bases code for the same amino acids.  However this does not mean that all species encode their genomes in exactly the same way.   
The code is ''redundant'': a number of triplets code for the same amino acid.  While all species are able to translate any sequence of DNA interchangeably, E. coli prefers to use certain triplets to code for certain amino acids which may be different to the ones we use.  This 'preference' is reflected in the levels of tRNA which match such a triplet.  In this project we resynthesised a number of genes ''de novo'' and thus were able to codon optimise them for expression in E. coli.
The code is ''redundant'': a number of triplets code for the same amino acid.  While all species are able to translate any sequence of DNA interchangeably, E. coli prefers to use certain triplets to code for certain amino acids which may be different to the ones we use.  This 'preference' is reflected in the levels of tRNA which match such a triplet.  In this project we resynthesised a number of genes ''de novo'' and thus were able to codon optimise them for expression in E. coli.
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[[Image:ribosome.jpg|300px|left|Ribosome translating mRNA]]
[[Image:ribosome.jpg|300px|left|Ribosome translating mRNA]]
Starting with the DNA sequence of the ''Vibrio fischeri'' lux operon found on the NCBI database, we used a number of tools to replace the codons used with the most common codons found in the E.coli genome. To achieve optimal expression of the Lux operon in E.coli, we had the operon re-synthesized after optimising the usage of codons. This conserves the sequence of amino acids in the gene products, but improves the rate of translation, as more common tRNAs are recruited. Codon usage optimization can yield dramatic increases in the expression of foreign genes, especially if they are introduced from less closely related species [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCW-4C8NKCY-3&_user=6094838&_coverDate=07%2F31%2F2004&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1511316646&_rerunOrigin=scholar.google&_acct=C000053194&_version=1&_urlVersion=0&_userid=6094838&md5=c970323b521fa1ec9d3050d4c4970eb1&searchtype=a Gustafsson et al. 2004]
Starting with the DNA sequence of the ''Vibrio fischeri'' lux operon found on the NCBI database, we used a number of tools to replace the codons used with the most common codons found in the E.coli genome. To achieve optimal expression of the Lux operon in E.coli, we had the operon re-synthesized after optimising the usage of codons. This conserves the sequence of amino acids in the gene products, but improves the rate of translation, as more common tRNAs are recruited. Codon usage optimization can yield dramatic increases in the expression of foreign genes, especially if they are introduced from less closely related species [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCW-4C8NKCY-3&_user=6094838&_coverDate=07%2F31%2F2004&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1511316646&_rerunOrigin=scholar.google&_acct=C000053194&_version=1&_urlVersion=0&_userid=6094838&md5=c970323b521fa1ec9d3050d4c4970eb1&searchtype=a Gustafsson et al. 2004]
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=Altered G-C content=
=Altered G-C content=
DNA curvature is increased by sequences rich in A-T or G-C pairs. The natural ''V.fischeri'' Lux operon, and especially its intergenic regions, contains stretches rich in A-T, resulting in the curvature that H-NS proteins bind to preferentially. Changing the coding DNA sequence also meant changing the curvature of the DNA, which affects the binding affinity of H-NS proteins. To alleviate the repression that H-NS exerts, we took care to raise the G-C content of intergenic regions and coding sequences (at times resorting to suboptimal codons). According to a computational prediction, this resulted in greatly reduced DNA curvature, and thus hopefully to a reduced affinity for H-NS proteins.
DNA curvature is increased by sequences rich in A-T or G-C pairs. The natural ''V.fischeri'' Lux operon, and especially its intergenic regions, contains stretches rich in A-T, resulting in the curvature that H-NS proteins bind to preferentially. Changing the coding DNA sequence also meant changing the curvature of the DNA, which affects the binding affinity of H-NS proteins. To alleviate the repression that H-NS exerts, we took care to raise the G-C content of intergenic regions and coding sequences (at times resorting to suboptimal codons). According to a computational prediction, this resulted in greatly reduced DNA curvature, and thus hopefully to a reduced affinity for H-NS proteins.
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[[Image:GC_content.png|600px|center| The LuxC coding region before and after codon optimisation. Blue denotes A-T rich regions. Note the reduction in A-T rich stretches after opitmisation]]
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{|
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|[[Image:GC_content.png|340px|left|G-C/A-T usage]]
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|[[Image:bending.jpg|340px|right|curvature of the Lux operon]]
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|-
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|'''Change in G-C(red)/A-T(blue) content by codon optimization. The occurence of long A-T rich stretches is reduced in the re-synthesised version of the Lux operon.'''
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|'''Computational prediction of DNA curvature before and after codon optimization. (click to enlarge)'''
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|}
=Differential Expression=
=Differential Expression=

Latest revision as of 00:13, 28 October 2010