Luciferase

Mutant luciferase (P. pyralis)

• Mutant Enhanced Photon Initiating Complex (EPIC) Luciferase (P.pyralis) Sequence
• Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color
• Improved practical usefulness of firefly luciferase by gene chimerization and random mutagenesis

Other Insect luciferases

• For colours we could use the click beetle luciferase red or green. They're reliable and from the Knight lab but not in the distribution.

Bacterial Bioluminescence

• Vibrio Fischeri
• Vibrio Harveyi
• Genetics of luxCDABE (adapted from wikipedia)
• 2006 attempts at BioBricking the lux operon
• Notes
• The lux wiki from parts registry
• NCBI Lux operon sequence
• parts registry lux operon -currently waiting on an email to see how useful/available this part is
• IMPORTANT INFO
• The Photorhabdus luminescens luxCDABE operon in the registry. It's not well characterised

Plasmid experiment

• Vibrio plasmid experiment -possible source of lux operon

File:4449975.pdf

• [1] - Transformation protocol for students from basics - includes several plasmids

• Plan
  • set plasmid 555 into a BioBrick
  • replace the promoter? sensitivity tuners?

Transformation Experiment to test Firefly luciferase in registry

• Firefly luciferase is in the registry as BBa_I712019, and have been sent to us as part of the spring 2010 distribution.

• We need to find a new vector with a bacterial ribosyme binding site, initiator, and a stop codon. Having searched via the catalog in the plasmids section, then in expression plasmids, we have selected a constitutive plasmid:
  • The plasmid selected is BBa_J13002, which is a TetR repressed POPS/RIPS generator, again part of the spring 2010 distribution - kit plate 1, well 13B, plasmid pSB1A2. It was proven to work by UMN iGEM 2009.

Wavelengths

548 nm http://departments.kings.edu/biology/lux/bacterial.html

613 nm http://www.promega.com/pnotes/85/10904_11/10904_11.pdf