Revision as of 11:10, 2 August 2010

Luciferase

[http://www.ncbi.nlm.nih.gov/nuccore/AB261988.1 Mutant Enhanced Photon Initiating Complex (EPIC) Luciferase (P.pyralis) Sequence]
[http://www.ncbi.nlm.nih.gov/pubmed/10529195 Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color]
[http://www.ncbi.nlm.nih.gov/pubmed/12044905 Improved practical usefulness of firefly luciferase by gene chimerization and random mutagenesis]

For colours we could use the click beetle luciferase [http://partsregistry.org/wiki/index.php/Part:BBa_J70005 red] or [http://partsregistry.org/wiki/index.php/Part:BBa_J70006 green]. They're reliable and from the Knight lab but not in the distribution.

Vibrio Fischeri
Vibrio Harveyi
Genetics of luxCDABE (adapted from wikipedia)
[http://parts.mit.edu/wiki/index.php/Lux_operon| 2006 attempts at BioBricking the lux operon]
Notes
[http://partsregistry.org/Lux The lux wiki from parts registry]
[http://www.ncbi.nlm.nih.gov/nuccore/AF170104.1?report=graph&log$=seqview NCBI Lux operon sequence]
[http://partsregistry.org/Part:BBa_G10001 parts registry lux operon] -currently waiting on an email to see how useful/available this part is
IMPORTANT INFO
The Photorhabdus luminescens [http://partsregistry.org/Part:BBa_J70008 luxCDABE operon] in the registry. It's not well characterised

[http://departments.kings.edu/biology/lux/bacterial.html Vibrio plasmid experiment] -possible source of lux operon

[http://departments.kings.edu/biology/lux/index.html] - Transformation protocol for students from basics - includes several plasmids

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

references
- [http://www.pnas.org/content/84/19/6639.short Overproduction and purification of the luxR gene product: Transcriptional activator of the Vibrio fischeri luminescence system (Kaplan & Greenburg 1987) ]
- [http://jb.asm.org/cgi/content/abstract/172/7/3974 Critical regions of the Vibrio fischeri luxR protein defined by mutational analysis (Slock et al 1990)]

Firefly luciferase is in the registry as [http://partsregistry.org/Part:BBa_I712019 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 [http://www.partsregistry.org/Part:BBa_J13002 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.

548 nm http://pubs.acs.org/doi/pdf/10.1021/bi7015052

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

@@ Line 38: / Line 38: @@
 **set plasmid 555 into a BioBrick
 **replace the promoter? sensitivity tuners?
+*references
+** [http://www.pnas.org/content/84/19/6639.short Overproduction and purification of the luxR gene product: Transcriptional activator of the Vibrio fischeri luminescence system (Kaplan & Greenburg 1987) ]
+** [http://jb.asm.org/cgi/content/abstract/172/7/3974 Critical regions of the Vibrio fischeri luxR protein defined by mutational analysis (Slock et al 1990)]
 ==Transformation Experiment to test Firefly luciferase in registry==