Difference between revisions of "Help:Standards/Assembly/RFC12"

Revision as of 09:04, 17 April 2012

Motivation & Discussion

The BioBrick BB-2 RFC[12] is a standard for interchangeable parts based on idempotent assembly. RFC[12] was designed to fix some concerns with BioBrick RFC[10]: the scar created between assembled parts is 6bp long and allows for in-frame protein assembly.

The BB-2 is a relatively recent standard and has not been adopted well enough within the synthetic biology community to fully address any possible concerns. This also means that while the Registry has parts that will be compatible with this assembly standard we do not have many available part samples that use this assembly standard. RFC[12] is incompatible with BioBrick RFC[10].

Advantages

• in-frame assembly of protein parts
• benign protein scar (Ala-Ser)
• N-end rule save
• introduction of only one new enzyme
• NheI is comparatively rare in the E. coli genome -- less background fragments from genomic DNA
• NheI can be heat-inactivated

Disadvantages

• incompatible to BioBrick RFC[10] format
• incompatible to Silver RFC[23] format

Technical Specifications

Compatability/Illegal Sites

In order for a part to be BioBrick BB-2 compatible it must not contain the following restriction sites, as these will need to be unique to the prefix and suffix:

• EcoRI site: GAATTC
• SpeI site: ACTAGT
• NheI site, GCTAGC
• PstI site: CTGCAG
• NotI site: GCGGCCGC

Additionally, there are a set of sites which, if convenient, should also be eliminated. Parts containing these sites qualify as fully Biobrick BB-2 standard compliant, but future assembly and advanced uses of the parts may be compromised. These include:

• PvuII site: CAGCTG
• XhoI site; CTCGAG
• AvrII site: CCTAGG
• XbaI site: TCTAGA
• SapI site: GCTCTTC and GAAGAGC

Prefix and Suffix

Image: Simple showing restriction sites

           Prefix                            Suffix
5' GAATTC GCGGCCGC ACTAGT ...part... GCTAGC GCGGCCG CTGCAG 3'
    EcoRI   NotI    SpeI              NheI    NotI   PstI 

Assembling two parts leaves the following scar:

5' [part A] GCTAGT [part B] 3'
             A  S

Primers

Prefix

5' GTTTCTT C GAATTC GCGGCCGC  ACTAGA ... part 3'
   (1)    (2)  (3)     (4)       (5)       (6)

1. Extra bases designed to both
   1. permit cutting of the PCR product with EcoRI by providing extra "spacer" bases.
   2. promote addition of an A base on the opposite strand by Taq polymerase for high efficiency TA cloning if desired.
2. Random extra spacer base
3. EcoRI recognition site
4. NotI recognition site
5. XbaI recognition site
6. Approximately 20 bp of sequence that matches the 5' end of the part you wish to construct.

Suffix

5' GTTTCTT C CTGCAG CGGCCGC GCTAGC ... part 3'
    (1)   (2)  (3)    (4)    (5)       (6)

1. Extra bases designed to both
   1. permit cutting of the PCR product with PstI by providing extra "spacer" bases.
   2. promote addition of an A base on the opposite strand by Taq polymerase for high efficiency TA cloning if desired.
2. Random extra spacer base
3. PstI recognition site
4. NotI recognition site
5. NheI recognition site
6. Approximately 20 bp of sequence that matches the end of the part you wish to construct.

Notes

Related Links

• Standards
• 3A Assembly
• Gibson Assembly

Sources

• [http://dspace.mit.edu/handle/1721.1/45139 Draft Standard for Biobrick BB-2 Biological Parts]
• [http://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/Formats#BBF_RFC_12:_Tom_Knight.27s_BB-2_proposal The BioBricks Foundation - Formats Discussion]