CH391L/S12/Restriction enzymes and BioBricks assembly standards

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Advancements in restriction enzymes)
(Advancements in restriction enzymes)
Line 38: Line 38:
Zinc finger (ZF) nucleases are artificial restriction enzymes.
Zinc finger (ZF) nucleases are artificial restriction enzymes.
-
The restriction enzyme FokI has two distinct domains: a DNA-binding domain, and a nuclease domain. The DNA-binding domain can be replaced with an engineered domain recognizing any sequence, allowing cleavage at any desired site. Additional site specificity comes from the fact that the ZF nuclease must dimerize in order to cleave the DNA, so two different binding sites must be specified that flank the site to be cleaved<cite>Kim1996</cite>.
+
The restriction enzyme FokI has two distinct domains: a DNA-binding domain, and a nuclease domain. The DNA-binding domain can be replaced with an engineered domain recognizing new sequences, allowing cleavage at a desired site. Additional site specificity comes from the fact that the ZF nuclease must dimerize in order to cleave the DNA, so two different binding sites must be specified that flank the site to be cleaved<cite>Kim1996</cite>.
==Commercial sector==
==Commercial sector==

Revision as of 19:53, 12 February 2012


Contents

Overview of restriction enzymes

What is a restriction enzyme?

A restriction enzyme, or restriction endonuclease, cleaves DNA at a restriction site.

Restriction enzymes are split into four types. Type I have multiple subunits and are not very useful because they randomly cleave the DNA distant from the recognition site, making the fragments produced unpredictable.

EcoRI's restriction site. Note the inverted-repeat symmetry.
EcoRI's restriction site. Note the inverted-repeat symmetry.
EcoRI's restriction site in molecular detail. Positive residues hold the backbone in place so that the recognition site can be recognized. Image from xray crystal structure of 1ERI[1].
EcoRI's restriction site in molecular detail. Positive residues hold the backbone in place so that the recognition site can be recognized. Image from xray crystal structure of 1ERI[1].

Type II are much more useful in the lab, because they cut DNA close to or within the recognition sequence (4-8 base pairs that exhibit inverted-repeat symmetry--see figure). In addition, type II endonucleases do not require energy from ATP hydrolysis[2].

An example type II restriction enzyme is EcoRI (E.C.3.1.21.4). The E.C. number specifies that this enzyme is a type II site-specific endodeoxyribonuclease producing 5'-phosphomonoesters.

For a step-by-step guide to cloning using restriction enzymes, see the Barrick Lab's wiki.

Digestion

After the digest reaction, the recognition site will be cleaved, with either blunt (no single-stranded DNA) or sticky (single-stranded overhang) ends. Isoschizomers recognize the same restriction site, but they can cleave in a different pattern—both blunt and sticky, with varying amounts of overhang[3]. The 5' phosphates remain attached, potentially allowing self-ligation, so a phosphatase step may be necessary following digestion.

Cleavage can be prevented by methylating the recognition sequence, on A or C nucleotides[3]. The host's DNA is methylated by methyltransferases, so that foreign DNA gets digested[2]. In this way, restriction enzymes can be thought of as a basic immune system.

Partial digestion occurs when the concentration of restriction enzymes is too low. This can be desirable, such as when there is more than one recognition sequence[3].

Advancements in restriction enzymes

Type IIS endonucleases

Type II vs. IIS endonucleases[4].
Type II vs. IIS endonucleases[4].

Type II restriction enzymes result in a scar when two fragments are ligated together. Type IIS endonucleases solve this problem by causing the sticky ends to be part of the gene itself, not the recognition sequence (see figure).

Zinc finger nucleases

Zinc finger (ZF) nucleases are artificial restriction enzymes.

The restriction enzyme FokI has two distinct domains: a DNA-binding domain, and a nuclease domain. The DNA-binding domain can be replaced with an engineered domain recognizing new sequences, allowing cleavage at a desired site. Additional site specificity comes from the fact that the ZF nuclease must dimerize in order to cleave the DNA, so two different binding sites must be specified that flank the site to be cleaved[5].

Commercial sector

New England Biolabs

New England Biolabs (NEB) is a prominent supplier of reagents for the biological sector. It places most of its focus on restriction endonucleases, offering over 260 different varieties. They also provide free use of REBASE, a restriction enzyme and methyltransferase database, and Polbase, a DNA polymerase database.

BioBrick/BglBrick assembly standards

Purpose of RFCs

RFCs (requests for comments) have multiple purposes, including describing standards/protocols, how-to descriptions, and referencing previous RFCs to extend them or make them obsolete.

Assembly 10: the original BioBricks assembly standard

Assembly standard 10 was introduced by BBF RFC 10 in 2003.

Making BioBrick parts

BioBrick parts can be constructed by adding the prefix and suffix to a part with PCR.

The prefix is prepended to a primer sequence:

5' GTTTCTTC GAATTC GCGGCCGC T TCTAGA G 3'
            <EcoRI> <NotI>    <XbaI>

Note that if the part codes for a protein, the prefix must instead be:

5' GTTTCTTC GAATTC GCGGCCGC T TCTAG 3'
            <EcoRI> <NotI>    <XbaI>

The suffix is:

5' T ACTAGT A GCGGCCG CTGCAG 3'
     <SpeI>  <NotI>    <PstI>

So the other primer would include the reverse complement of this sequence:

5' GTTTCTTC CTGCAG CGGCCGC T ACTAGT A 3'
            <PstI>  <NotI>   <SpeI>

After PCR, these parts will have the standard triplet of restriction sites that make up the BioBricks prefixes and suffixes.

Putting two BioBrick parts together

Standard assembly under RFC 10 (image from PartsRegistry.org).
Standard assembly under RFC 10 (image from PartsRegistry.org).

Ligating two BioBrick parts together results in a scar between the two parts: because the suffix restriction site does not match the prefix restriction site, upon ligation the palindrome is broken and will no longer comprise either recognition sequence. The scar codes for a tyrosine residue and a stop codon, plus two additional nucleotides (5' AG 3') that cause a frame shift. This means that fusing two proteins together cannot be done under Assembly 10, a significant disadvantage, driving the development of new RFCs that allow protein fusion. These standards include BBF RFC 23 ("Biofusion", which makes the scar into a 6-bp sequence that codes for tyrosine and arginine, with no extra nucleotides to cause a frame shift), BBF RFC 25 ("Fusion parts", which also eliminates the frame shift but changes the second residue from the problematic arginine to a glycine), and BBF RFC 21 ("Berkeley format" AKA BBb AKA BglBricks).

Assembly 21: BglBricks

Assembly 21 is described by BBF RFC 21. The main difference is its use of a different set of isocaudomers (restriction enzymes that recognize slightly different sequences but result in the same sticky ends after digestion), BamHI and BglII instead of XbaI and SpeI. It also uses XhoI instead of PstI, and NotI is not included.

Advantages of BglBricks

Assembly under RFC 21[6].
Assembly under RFC 21[6].

Parts adhering to RFC 21 can be fused together without a frame shift. In addition, the 6-bp scar between parts after fusion is a relatively safe glycine-serine sequence, without the stop codon present in RFC 10. Finally, the restriction enzymes have a higher efficiency than those used in RFC 10[7].

Disadvantages of BglBricks

BglBricks are incompatible with RFC 10.


References

  1. Kim YC, Grable JC, Love R, Greene PJ, and Rosenberg JM. . pmid:2399465. PubMed HubMed [Kim1990]
    Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.

  2. Pingoud A and Jeltsch A. . pmid:11557805. PubMed HubMed [Pingoud2001]
    Structure and function of type II restriction endonucleases.

  3. Bloch, Kenneth and Grossmann, Barbara. "Restriction Endonucleases." Current Protocols in Molecular Biology (1995) 3.1.1-31.21 [Bloch1995]
  4. Lippow SM, Aha PM, Parker MH, Blake WJ, Baynes BM, and Lipovsek D. . pmid:19304757. PubMed HubMed [Lippow2009]
    Creation of a type IIS restriction endonuclease with a long recognition sequence.

  5. Kim YG, Cha J, and Chandrasegaran S. . pmid:8577732. PubMed HubMed [Kim1996]
    Hybrid restriction enzymes: zinc finger fusions to FokI cleavage domain.

  6. Anderson JC, Dueber JE, Leguia M, Wu GC, Goler JA, Arkin AP, and Keasling JD. . pmid:20205762. PubMed HubMed [Anderson2010]
    BglBricks: A flexible standard for biological part assembly.

  7. http://partsregistry.org/Help:Assembly_standard_21 [RFC21]
All Medline abstracts: PubMed HubMed
Personal tools