Team:Cambridge/Bioluminescence/Colour

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Revision as of 19:43, 26 October 2010

Project Firefly: Coloured outputs

The peak emission wavelength of luciola cruciata luciferase can be altered by single amino acid changes as reported by Kajiyama and Nakano. Different coloured outputs, as well as being aesthetically pleasing, allow the most appropriate wavelength of output for a particular detector to be chosen. By putting different coloured luciferases under promoters which respond to different inputs, co-reporter assays would be possible.

Comparison of the colour of Luciola cruciata we ordered from DNA 2.0 and it's colour after a single amino acid substitution

Methods

We used site directed mutagenesis to produce these specific mutant luciferase genes. From DNA 2.0 we ordered the sequence of a red-shifted mutant with a Ser286Asn codon change. The luciferase produces an orange glow as shown. The construct was then mutated back to the wild type gene then different point mutations were introduced. pSB1C3 with the luciferase and LRE genes is a sizeable plasmid so we performed two separate PCR reactions to generate the mutagenised construct in two halves (using oligonucleotides with the point mutation). PCR products were run on an agarose gel and the relevant bands gel extracted then joined by Gibson Assembly. Cells were transformed with the product. This method avoids cells being transformed with the non-mutated, template plasmid. As an additional check colonies which grew from successfully transformed cells were imaged inside a dark box after addition of luciferin to check the colour of the light produced was as expected.

Results

A co-reporter system

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 ==Methods==
-We used site directed mutagenesis to produce these specific mutations. pSB1C3 with the luciferase and LRE genes is a sizeable plasmid so we performed two separate PCR reactions to generate two halves of the plasmid (using oligonucleotides with the point mutation). PCR products were run on an agarose gel and the relevant bands gel extracted then joined by Gibson Assembly. Cells were transformed with the product. This method avoids cells being transformed with the non-mutated, template plasmid. As an additional check colonies which grew from successfully transformed cells were imaged inside a dark box after addition of luciferin to check the colour of the light produced was as expected.
+We used site directed mutagenesis to produce these specific mutant luciferase genes. From DNA 2.0 we ordered the sequence of a red-shifted mutant with a Ser286Asn codon change. The luciferase produces an orange glow as shown. The construct was then mutated back to the wild type gene then different point mutations were introduced. pSB1C3 with the luciferase and LRE genes is a sizeable plasmid so we performed two separate PCR reactions to generate the mutagenised construct in two halves (using oligonucleotides with the point mutation). PCR products were run on an agarose gel and the relevant bands gel extracted then joined by Gibson Assembly. Cells were transformed with the product. This method avoids cells being transformed with the non-mutated, template plasmid. As an additional check colonies which grew from successfully transformed cells were imaged inside a dark box after addition of luciferin to check the colour of the light produced was as expected.
 ==Results==