BU06:Research

From 2006.igem.org

Revision as of 00:00, 16 June 2006

Making E. coli Glow

Preliminary Design: -Light Sensing Inhibitor- -> -Light emission device-

Light Sensing Inihibitor: [http://partsregistry.org/Part:BBa_I15008 BBa_I15008] , [http://partsregistry.org/Part:BBa_I15009 BBa_I15009] , [http://partsregistry.org/Part:BBa_I15010 BBa_I15010] , [http://partsregistry.org/Part:BBa_R0082 BBa_R0082]

Light Emission Device: (from lux [http://en.wikipedia.org/wiki/Operon operon]) THIS IS WHAT WE NEED TO DO.

Lux Operon

Point Mutation: Lamda Red - Datsenko, Wanner... or by PCR: look for protocol on OpenWetWare\

Codon Usage Bias Database: http://www.kazusa.or.jp/codon/

Light Sensor Parts: http://partsregistry.org/Featured_Parts:Light_Sensor and paper : http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html (this paper simply explains how the device works!)

It seems to repress gene expression by having red light inhibit phosphorylation which would activate a promoter. We would replace the LacZ protein coding region with our lux [http://en.wikipedia.org/wiki/Operon operon].

Part: BBa_F1610 codes for LuxI, should we need it...

Sequence details:

[http://www.ai.mit.edu/projects/ntt/documents/proposals2000/MIT9904-10/proposal.html According] to Tom Knight, the Photorhabdus luminescens luxCDABE operon that he cloned is NCBI accession number [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=155405 M90093]. I checked this sequence against the biobrick restriction enzymes (EcoRI, XbaI, SpeI, PstI, NotI) using the [http://bioinformatics.org/sms2/rest_summary.html Sequence Manipulation Suite]. Results: EcoRI cuts at the ends of the sequence (+2 and -4; i.e., the original sequence is intended to be cut out of its vector with EcoRI); XbaI cuts in the middle (+2411); and SpeI, PstI, and NotI do not cut M00093. The question therefore becomes, did Tom Knight's group add or remove restriction sites? We have the DNA, we can test this in lab.

Silent Point Mutation: XbaI at bp 2411 of P. luminescens LuxCDABE

                _/_____       <---XbaI cut
           5' AAT CTA GAT 3'
               N   L   D
              Asn Leu Asp

          Triplets for these A.A.'s
               N    L   D
              AAT  TTA GAT
              AAC  TTG GAC
                   CTG
                   CTT
                   CTC
                   CTA

          Codon Usage Bias! (Frequency: per thousand) 
          Leu Codon     E.coliK12      P.lumin
             CUU           11.0          12.1
             CUC           11.0           9.1
             CUA            3.9           9.0
             CUG           52.8          29.6

CUA in P.lum is 9.0/1000, for a silent point mutation of Leucine, CUU or CUC with frequency of 11.0/1000... and its still Leu!!!!

Brainstorming

• could we print LB + bacteria onto paper with an inkjet?
• what about printing the AHL quorum signals? Or other chemicals that could spatially control E. coli or compel them to do something (i.e. cause them to "print" or digest a substrate)?
• If we are successful with our light-repressed luminesence system, to what civic uses could we put it? Pathway / sidewalk lighting?
• What about re-engineering E. coli to repair things... such as fading paint on a building? I guess that's just like bioremediation.
• This is really wild: what if we understood the process from stem cell to tooth so well that we could predict and remodel the final structure using computers and then reprogram the stem cells for biofabrication? It wouldn't exactly be rapid prototyping, but it would be massively cheap!
• I know people have transformed the membrane oscillations of yeasts into [http://www.darksideofcell.info/ audible sounds] (using an atomic force microscopes) and I wonder if E. coli could be signalled or manipulated in some way with acoustic waves.

--Mac 00:35, 14 June 2006 (EDT)