Difference between revisions of "CH391L/S14/BioBricks"

Revision as of 20:40, 18 January 2014

What are BioBricks, and what is their purpose?

A BioBrick is a sequence of DNA with a predefined structure and function. The information is held in a plasmid, or a circular piece of DNA that can be inserted, and replicated into bacteria.

Before BioBricks, there was little standardization in assembly techniques for DNA sequences. It was necessary to create a standardized system of biological building blocks to enable scientists to go beyond the experiment of DNA assembly and allow them to focus on more complicated research. BioBricks were created as a set of standard and interchangeable “parts” that could be assembled into sub-components. According to the Registry of Standard Biological Parts, which contains DNA for thousands of previously created parts submitted by iGEM members, a standard biological part is “a functional unit of DNA that encodes for a specific biological function. Parts have been standardized so they can be used to efficiently develop biological systems in living cells." The advantage of BioBricks is that the assembly of these parts could be outsourced to others and new experimentation could rely heavily on previously manufactured components. The BioBricks foundation was co-founded in 2005 by Drew Endy. Tom Knight and Drew Endy also helped co-found the Registry of Standard Biological Parts.[1]

The creation of a standard sequence interface for all BioBricks:

The original BioBrick assembly standard created by Tom Knight:

In order to easily joint and manipulate segments of DNA, BioBrick assembly standards requires the use of defined prefix and suffix sequences that flank both sides of the BioBrick. These prefix and suffix regions (such as EcoRI, NotI, XbaI, SpeI, and PtsI) can be cut by specific restriction endonucleases. Restriction endonucleases are a key element in the cutting and conjoining of segments of DNA, and will be elaborated in further detail in a later section. (insert hyperlink here.. ask dennis how to link it to restriction endonuclease section)

The upstream end (Prefix) contains vector insert restriction sites with the following sequence:

5' --gca GAATTC GCGGCCGC T TCTAGA G --- 3'
3' --cgt CTTAAG CGCCGGCG A ACATCT C --- 5'
         EcoRI    NotI       XbaI

The downstream end (Suffix) contains vector insert restriction sites with the following sequence:

5' --- T ACTAGT A GCGGCCG CTGCAG gct--- 3'
3' --- A TGATCA T CGCCGGC GACGTC cga--- 5'
          SpeI     NotI     PstI

When combined, the entire component vector looks like this:

 5’ --gca GAATTC GCGGCCGC T TCTAGA --insert--T ACTAGT A GCGGCCG CTGCAG gct--
 3' --cgt CTTAAG CGCCGGCG A ACATCT --------  A TGATCA T CGCCGGC GACGTC gca--
          EcoRI     NotI     XbaI               SpeI     NotI     PstI

The component vector (the insert) cannot contain any of the restriction sites to avoid any unwanted cutting by restriction enzymes, and any such sites are removed by point mutations.

Restriction Endonucleases

Restriction enzymes, or restriction endonucleases are enzymes that cleave DNA at restriction sites. A restriction site is a sequence of base pairs (generally 4-8 base pairs in length) that is recognized and spliced by a restriction enzyme. To cut DNA, restriction enzymes make two incisions: once through each side of the sugar-phosphate backbone. There are 5 kinds of restriction enzymes.

Types of Restriction Endonucleases

Type I: These were the first type of enzymes to be discovered, but have very little use in synthetic biology because they randomly cleave the DNA varying distances from the restriction site. The recognition site is composed of two different, asymmetrical portions: one containing 3-4 nucleotides, and another containing 4-5 nucleotides, and are separated by a non-specific spacer that is composed of 6-8 nucleotides.

Type II: These are the most commonly available and used restriction enzymes. They are a homodimer, meaning that they are composed of two identical molecules, and they use no ATP for their activity. Type II restriction enzymes recognize palindromic sequences, which are sequences that are read the same 5’ as they are 3’. New enzymes from this family have been discovered, but do not follow all classical criteria of type II enzymes. Subgroups have been created to categorize the enzymes that deviate from the criteria.