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Construction of Short Parts

So far we've dealt with the scenario in which your part exists in some source DNA. Either you are amplifying your part sequence from genomic DNA, cDNA, or perhaps a plasmid someone gave you as a gift. There are a number of scenarios in which you might want to use a different source for your parts:

Gene Synthesis

Sometimes you don't have the source DNA. Perhaps the sequence comes from a source organism that is not available to you. Perhaps it is a eukaryotic CDS and you wish to express it in a bacteria. In that case, you might want to change the codon usage and eliminate all the intron sequences. If you have a sequence that is over 120bp or so, and you can't simply amplify the sequence from some source DNA, your only option is Gene Synthesis. There are a number of commercial suppliers who will synthesize your part and send you plasmid DNA (but it's pretty expensive). You can also do gene synthesis on your own. There is a website called GeneDesign that will help you design your part and determine the oligos you should order to assemble your part.

Overlap Extension

Suppose your part is between 30bp and 120bp. Even if you have a source DNA for the part, the best way to construct it is by overlap extension (also called a Klenow extension, or as we call it in Anderson lab, a wobble reaction). In an overlap extension, you construct 2 oligos that are reverse complementary to one another over 20bp on their 3' ends. There is no template DNA in the reaction--you simply combine the two oligos in one reaction, anneal them to one another, and then fill in the rest of the fragment using a polymerase. Traditionally such reactions were done with the Klenow fragment of E. coli DNA polymerase I. Today, the most effective way to do it is with a thermostable polymerase.

To design the oligos, start by putting your part including the flanking restriction sites into ApE. As an example, let's make a part encoding the Ala2 tRNA:


So, put that sequence into ApE and then add the EcoRI, BamHI, and BglII sites:


Also add some 5bp tails to the ends:


Now, identify a 20bp sequence that limits secondary structure, has a good GC balance, low repetitive sequence (the usually rules for designing a good annealing region). Copy it to your clipboard, ctrl-F to find, search for the sequence and highlight all. In this case, I've chosen:


To design your oligos, copy the sequence from the 5' end through to the end of the highlighted region. This is your forward oligo:


Now grab the sequence from the beginning of the highlighted region through to the 3' end of the sequence and reverse complement it:


That's it! Just write up the construction file and you are done:

 Wobble ca9939/ca9940           (107bp, EcoRI/BamHI)
 Sub into pBca9145-Bca1144#5    (EcoRI/BamHI, 2057+910, L)
 Product is pBca9145-Bca9939     {Ala2}
 ca9939   Forward construction of Ala2 basic part
 ca9940   Reverse construction of Ala2 basic part