BU06:Team D

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Team Goal: 'Mutagenesis of XbaI

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Meeting Schedule

Current Progress

Note: 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 operon(makes light!) from Photorhabdus luminescens M90093 into a BioBrick.

Hopefully, you realize (from the readings), that BioBricks have standard plasmid constructs with 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 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 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 codon bias as P. luminescens. We can change CTA to CTU or CTT. Check for yourself at 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 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 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
          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!!!!



=== Protocols ===

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