Team:Cambridge/Bioluminescence/Colour

smith and smith reported point mutations that change the colour of luciola cruciata luciferase. Different coloured outputs would allow co-reporter assays and

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 sucessfully transformed cells were imaged inside a dark box after addition of luciferin to check the colour of the light produced was as expected.

Results

We produced...

BBa_K325210

Luciola cruciata LRE and luciferase

This translational unit combines the luciferin regenerating enzyme (BBa_K325202) and red-shifted luciferase (BBa_K325211) from the Japanese firefly, Luciola cruciata. The luciferin regenerating enzyme helps to strengthen and sustain light output.
BBa_K325100

Photinus pyralis LRE and luciferase

This part combines the luciferin regenerating enzyme (BBa_K325102) and luciferase (BBa_K325101) from the North American firefly, Photinus pyralis. The luciferin regenerating enzyme helps to strengthen and sustain light output.
BBa_K325109

Photinus pyralis LRE and luciferase under pBAD

This part is the part above expressed under pBAD for characterisation.
BBa_K325209

L. cruciata LRE and luciferase (wildtype) under pBAD

This part combines the Luciferin Regenerating Enzyme and luciferase from the Japanese firefly, Luciola cruciata. It is expressed under pBAD.
BBa_K325229

L. cruciata LRE and luciferase (green) under pBAD

This is the same as the part above but with a Val239Ile mutation which green-shifts its emission spectrum.
BBa_K325239

L. cruciata LRE and luciferase (red) under pBAD

This is the same as the part above but instead has a Gly326Ser mutation which red-shifts its emission spectrum.
BBa_K325249

L. cruciata LRE and luciferase (scarlet) under pBAD

This is the same as the part above but instead has a His433Tyr mutation which red-shifts its emission spectrum.
BBa_K325259

L. cruciata LRE and luciferase (yellow) under pBAD

This is the same as the part above but instead has a Pro452Ser mutation which shifts its emission spectrum to peak at a yellow wavelength.