Help:Standardization

Revision as of 17:38, 23 June 2006 by Smelissali (Talk | contribs) (→‎Checking for Restriction sites)

Starting out

What you want: A Biobrick standardized sequence of interesting DNA

So I've know of this interesting gene...
Find that sequence! This can be a lot harder than it sounds...but make sure you can find out the following specifics:

  • What strain is it in? Is it difficult to work with in the lab?
  • How well studied is the gene?

A good place to start looking for sequence is [http://www.ncbi.nlm.nih.gov/Genbank/index.html NCBI's Genbank]

What you've got now: A non-Biobrick-standardized sequence of DNA that does something interesting

So here's how you do it...

Checking for Restriction sites

  • First off, check your DNA sequence for the presence of Biobricks restriction sites. This is automatically done when you Add a part! Just enter the sequence of your part, and the Registry will give you the base pair coordinates of where unwanted restriction sites are in your part.

What you've got now:A piece of sequenced DNA with identified Biobricks restriction sites that you need to get rid of!

Removing the Biobricks Restriction sites on your sequence

  • Next, locate and remove all of the BioBrick standard restriction sites (XbaI, SpeI, EcoRI, PstI) that are on your sequence of interest. To remove these sites, these are some popular procedures:
    1. site directed mutagenesis - best for changing small, targeted areas of the genome like the Biobricks standard restriction sites. Primers with the desired mutation align to the area you would like to change, and then PCR amplification extends them. Openwetware has a nice [http://openwetware.org/wiki/Site-directed_mutagenesis protocol on how to do this], and there's more information in this [http://www.stratagene.com/manuals/200518.pdf Stratagene Mutagenesis Protocol (PDF download)]
    2. lambda red - best for if you have very large (thousands of kb, entire genes) areas for alteration or deletion. This process is laid out in [http://www.pnas.org/cgi/content/full/97/12/6640 Datsenko and Wanner, 2000]

What you've got now: A cleaned-up area of sequence with no Biobricks restriction sites in it

Adding BioBrick Ends

  1. If your gene is small enough....Order primers with the Biobricks end sequences (available in Part:BBa_G00000 and Part:BBa_G00001) according to Standard Assembly and PCR out the results
  2. If your gene is large...We recommend getting your gene synthesized, but be forewarned, this can be a costly process with issues about intellectual property (more on this later...)

What you've got now: Pieces of DNA with Biobrick ends in solution ready to be ligated and transformed into a containing plasmid vector


Links

  • [http://parts.mit.edu/wiki/index.php/BU06:Research The BU iGEM team] is working on standardizing the Lux operon into a Biobrick using these techniques with a really spirited walkthrough and log of their progress