BU06:Team D
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| Team Goal: 'Mutagenesis of XbaI |
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Contents |
Meeting Schedule
Current Progress
Note: [http://www.stratagene.com/products/showProduct.aspx?pid=505 Stratagene QuikChange® XL Site-Directed Mutagenesis Kit]
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!
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!!!!
References
Techniques
=== Protocols ===
