Haynes:BioBrick Method short

From OpenWetWare
Revision as of 14:02, 11 September 2012 by Karmella Haynes (talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to: navigation, search

<- Back to Protocols

Assembly of BioBrick Parts: an Overview

By Karmella Haynes, 2012

If you are new to molecular biology, please refer to a general biology reference (college biology text book, Wikipedia, etc.) or click the "ref" link for more details on the terms in bold print.

A BioBrick is a standardized piece of DNA that is flanked by a set of restriction enzyme cut sites (ref), e.g., EcoRI (E), NotI (N), XbaI (X) upstream (left); SpeI, NotI, PstI downstream (right). What makes the DNA a "standard part" is that the users within a community agree to flank their DNA fragments with the same sites, and they make sure that the cut sites do not appear within their DNA parts, so that parts can be shared freely without the time-consuming process of figuring out a new strategy every time a user wants to assemble a synthetic gene. The standard shown below is RFC23, or the "Silver Standard" developed by Pam Silver's lab at Harvard:

gaattc gcggccgc a tctaga [BioBrick Part] actagt a gcggccgctgcag
  EcoRI   NotI       XbaI                   SpeI     NotI   PstI  
BioBrick Prefix                       BioBrick Suffix

Note: At the BioBrick Suffix, the NotI and PstI cut sites overlap at the underlined “c.”

The BioBrick itself must not contain any of the standard restriction sites (internally), or else it will be cut apart during the assembly process. Basic BioBrick assembly uses one of the two strategies:

Ligating a “front insert” in a “front vector” to make A+B

Haynes BioBrickAssembly1.png

Ligating a “back insert” in a “back vector” to make the same A+B construct

Haynes BioBrickAssembly2.png

Note: After DNA plasmids are cut, the desired DNA fragments are isolated and purified using agarose gel electrophoresis (ref). The fragments that are bordered by a dotted line are discarded.

A few important words about DNA assembly: In the diagram above, all of the DNA starts out as a plasmid (circular piece of DNA). Each plasmid has a region called a backbone (shown as a thin arc) that consists of the origin of replication (the DNA sequence that encodes information for making copies of the plasmid; the mechanism is very complex) and an antibiotic resistance gene (e.g., ampicillin resistance). After the plasmids are cut with restriction enzymes (ref), one DNA fragment is used as an insert (no backbone) and another is used as the vector (has the backbone). The final DNA ligation product (the recombinant plasmid) must have one backbone.

About the "S/X" scar sequence: When two BioBricks are ligated, the SpeI end ligates with the XbaI end. The resulting sequence is a mixed site (called a scar) that cannot be re-cut by any enzyme. Silver Standard assembly produces a 6 b.p. scar (two codons) so that proteins can be fused together without creating a frame-shift mutation.