Help:Standardization

Revision as of 15:10, 20 December 2011 by Kitwa (Talk | contribs) (→‎Advantages of adding your part to a BioBrick Vector, before all the BioBricks Restriction sites are removed)

Starting out

What you want: A BiobBrick-standardized sequence of interesting DNA

So I 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 organism and strain is it in? Is it difficult to work with in the lab?
  • How well studied is the gene?
  • How long is the sequence? Is it so long (>10 kb) that it might cause problems?
  • What is the A-T:G-C content? If the ratio is skewed badly to one side, it can cause trouble with techniques like PCR assays (because of a very high annealing temperature, for example).

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 standardize 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 unwanted restriction sites in your part.

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

Advantages of adding your part to a BioBrick Vector, before all the BioBricks Restriction sites are removed

It often makes sense to add your part directly to a BioBrick vector before it is converted to a BioBrick part (and if your part does not contain either EcoI and SpeI sites or XbaI and PstI sites, then this step is not hard.) Adding your part to BioBrick vector before the restriction sites are removed has the advantage then you will be able to use the standard BioBrick sequencing primers VF2 and VR (parts BBa_G00100 and BBa_G00101) to sequence your part during site-directed mutagenesis and tools within the registry (see Physical DNA section to find the sequencing tools, [1] for some problems with using the primers, and [2] for a description of how the sequencing tools work) to keep track of your sequencing results and progress in converting your part to a BioBrick part.

It soon may even be possible to add your part to a BioBrick vector before all the restriction sites are removed, even if it contains all four BioBrick restriction enzyme recognition sites using a new type of BioBrick called BioScaffold parts [3]. As this capability becomes available, this page will be linked to protocols describing how to use BioScaffold parts to insert your non standard part into a BioBrick vector.

Removing the Biobricks Restriction sites on your sequence

  • Next, locate and remove all of the BioBrick standard restriction sites (XbaI, SpeI, EcoRI, PstI) that occur in your sequence of interest. Here are some popular procedures to remove these sites:
    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 stretch of sequence with no BioBricks restriction sites in it.

Adding BioBrick Ends

Information, specifics, and sequences of the characterisic BioBrick Prefix and Suffix can be found here.

  1. If your gene is small enough... Order primers with the BioBricks end sequences (available in Part:BBa_G00000 and Part:BBa_G00001) as well as the aformentioned page on prefix and suffix sequence according to Standard Assembly and use PCR to generate the desired BioBrick part.
  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 into a plasmid vector that can then be transformed into a suitable host cell.

Links

  • [http://2006.igem.org/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