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 only at the 'planning' stage though.

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?

• references
  • Overproduction and purification of the luxR gene product: Transcriptional activator of the Vibrio fischeri luminescence system (Kaplan & Greenburg 1987)
  • Critical regions of the Vibrio fischeri luxR protein defined by mutational analysis (Slock et al 1990)

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://pubs.acs.org/doi/pdf/10.1021/bi7015052

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