BU06:Research

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Wondering what's going on with Research??? Quick Overview!! (6/15/06)

Mostly, we've been concentrating on turning the LuxCDABE [http://en.wikipedia.org/wiki/Operon operon](makes light!) from Photorhabdus luminescens [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=155405 M90093] into a BioBrick.

Hopefully, you realize (from the [http://2006.igem.org/wiki/index.php/BU06:Reading_and_Discussion_1 readings]), that BioBricks have standard plasmid constructs with [http://partsregistry.org/cgi/htdocs/Assembly/index.cgi four specific restriction enzyme sites]. So, to turn a sequence into a BioBrick, it is essential that the sequence does not contain any of these restriction enzyme sites (or the digest would cut our BioBrick up!). You can use [http://tools.neb.com/NEBcutter2/index.php NEB Cutter] to check the sequence(link above) for these enzymes yourself! It turns out XbaI(EX-ba-wun), is located smack dab in the middle.. oh craps. We gotta do something about that.

What can we do??? One option is to do a [http://en.wikipedia.org/wiki/Point_mutation single point mutation], and change one base pair in the sequence so that XbaI no longer recognizes that site. The best option is to do a silent point mutation, where the changed base pair still results in the same amino acid. Looking at the sequence(data is below), there is a viable silent point mutation in the middle of the site with a similar [http://en.wikipedia.org/wiki/Codon_usage_bias codon bias] as P. luminescens. We can change CTA to CTU or CTT. Check for yourself at [http://www.kazusa.or.jp/codon/ Codon Bias Database]; Compare E. Coli K12 with P. luminescens

AWESOME!!! ...Now what?? We need your help! We need to know HOW to do POINT MUTATIONS! Under [http://2006.igem.org/wiki/index.php/BU06:resources links & resources], you will find steps on how to perform various bioengineering techniques under PROTOCOLS! We need to look up some protocols on point mutations and mutagenesis! Try looking at [http://www.stratagene.com/manuals/200518.pdf Stratagene Mutagenesis Protocol (PDF download)].

Whew! So that's where we are right now!

Adding Ends PCR! We will use the AflI restriction site in the middle where two PCR reactions will overlap and we can stick em together!

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!

Re: from what i thought, the understanding of stem cell lineage is not very well documented, with lots of discrepencies, and laboratory techniques to recreate them are few and very difficult! i may be wrong tho...

• 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)