Help:Assembly standard 15
Bioscaffold sites are BioBrick parts that contain restriction enzyme recognition sites that enable full or partial removal of the Bioscaffold site from a composite BioBrick part. Bioscaffold parts address some of the limitations of BioBrick standard assembly by enabling protein fusions, cloning of part libraries within a composite BioBrick part, and removal of BioBrick scars.
BioScaffold parts
The enzymes that facilitate the removal of BioScaffold parts are defined by what region of the part that they excise:
- External Enzymes: Do not cut solely inside the BioBrick Part (extend to Prefix, Suffix, and/or Scar regions)
- Internal Enzymes: Cut inside the BioBrick Part (between the Prefix, Suffix, and/or Scar regions)
The classes of α, β, and γ BioScaffold parts are defined for assemblies of the form:
<Prefix> part1 <Scar> BioScaffold part <Scar> part2 <Suffix>
- Class α parts: specific enzymes cut outside of scars surrounding the BioScaffold part
- Class β parts: specific enzymes cut within scars surrounding BioScaffold part
- Class γ parts: no specific enzymes, internal enzymes cut within the BioScaffold part
- Mixed parts: contain properties of several classes, name classes involved
Compatibility of plasmid backbones with BioScaffold sites
For a plasmid backbone to be compatible with BioScaffold sites, remove the following restriction enzymes.
- PpiI
- PsrI
- AarI
- MabI.
Also note that
- XmaI is being considered as an additional site for inclusion in BioScaffold sites.
- AscI and PacI are sometimes used instead of MabI because their restriction site sequences make them orthogonal to many parts based on GC content.
Contributing to the BioScaffold part collection
To join the BioScaffold community, you can construct and test existing BioScaffold part designs, design new BioScaffold parts, design new uses for BioScaffold parts, or convert high use plasmids to a BioScaffold compatible format.
If you are interested joining the community or have already done so, please contact Julie Norville at (julie dot norville at gmail dot com and norville at mit dot edu) with the subject heading "BioScaffold parts" so that your work and insights can be described in future BioScaffold parts BBF RFCs or publications and/or you can be solicited as a co-author on these documents.
You may also wish to describe your intent to test already designed BioScaffold parts or convert BioBrick vectors to the BioScaffold format on the BioScaffold Part User Group wiki [http://openwetware.org/wiki/BioScaffold_Parts_Users_Group BioScaffold Parts User Group wiki] (especially if you wish to convert plasmids or other important parts to a BioScaffold part compatible format, since this is a task that a number of users may contemplate) so that others in the community do not duplicate your efforts. As the community grows, we will create a mailing list.
Plasmid backbone compatibility with BioScaffold Part Internal and External Enzymes
Currently pSB2K4 is the only vector compatible with all the BioScaffold enzymes. Thus if you plan to do BioScaffold part operations, you may wish to consider using pSB2K4. If you wish to help make BioScaffold parts more useful to the community then you could consider converting some of the classic BioBrick vectors (for example pSB1AC3, pSB1AK3, pSB1AT3) into forms compatible with the BioScaffold parts. If you plan to do this, please inform Julie Norville (at julie dot norville at gmail dot com) so that your efforts can be included in future publications on BioScaffold parts.
| Plasmid | Incompatible Enzymes (# of Sites-priority for removal where H=high, M=medium, L=low, V=very low) | Compatible Enzymes | Commonly Used Plasmid |
|---|---|---|---|
| pSB1A3, discontinued [1] | PpiI(2, V) | AarI, AscI, BamHI, MabI, PacI, PsrI, XmaI | No |
| pSB1AC3, high copy [2] | PpiI(2, H) | AarI, AscI, BamHI, MabI, PacI, PsrI, XmaI | Yes |
| pSB1AK3, high copy [3] | PpiI(2, H) | AarI, AscI, BamHI, MabI, PacI, PsrI, XmaI | Yes |
| pSB1AT3, high copy [4] | PpiI(2, H), BamHI(?,M) | AarI, AscI, MabI, PacI, PsrI, XmaI | Yes |
| pSB1A7, transcriptionally insulated high copy [5] | PsrI(2, L) and PpiI(2, L) | AarI, AscI, BamHI, MabI, PacI, XmaI | Sometimes |
| pSB2K3, use pSB2K4 instead | AscI(1, V) | AarI, BamHI, MabI, PacI, PpiI, PsrI, XmaI | No |
| pSB2K4, inducible copy [6] | BamHI(?, L) | AarI, AscI, MabI, PacI, PpiI, PsrI, XmaI | Yes |
| pSB3C5, low to medium copy [7] | PpiI(1, L) | AarI, AscI, BamHI, MabI, PacI, PsrI, XmaI | Yes |
| pSB3K3, probably discontinued | PpiI(1, V), XmaI(?, V) | AarI, AscI, BamHI, MabI, PacI, PsrI | No |
| pSB3T5, low to medium copy [8] | PpiI(1, M) and PsrI(1, M) | AarI, AscI, BamHI, MabI, PacI, XmaI | Yes |
| pSB4A3, discontinued [9] | PpiI(1, V) | AarI, AscI, BamHI, MabI, PacI, PsrI, XmaI | No |
| pSB3K5, low to medium copy standard vector [10] | PpiI(1, M) | AarI, AscI, BamHI, MabI, PacI, PsrI, XmaI | Yes |
| pSB4C5, low copy standard vector [11] | None | AarI, AscI, BamHI, MabI, PacI, PpiI, PsrI, XmaI | Yes |
| pSB4K5, low copy standard vector [12] | None | AarI, AscI, BamHI, MabI, PpiI, PsrI, XmaI | Yes |
| pSB4A5, low copy standard vector [13] | PpiI(1, H) | AarI, AscI, BamHI, MabI, PacI, PsrI, XmaI | Yes |
| pSB4T5, low copy standard vector [14] | PsrI(1, H) | AarI, AscI, BamHI, MabI, PacI, PpiI, XmaI | Yes |
Enzymes used in BioScaffold standard
| Enzyme | Internal, External, BioBrick Enzyme | Typical Use | Special Property | Priority for Removal from Vector or Part | Digestion temperature | Heat killable (for automated assembly) | Efficiency | Buffers |
|---|---|---|---|---|---|---|---|---|
| AarI | External | Library Vector or Insert Creation, Rapid prototyping | Good for cutting into BioBrick scars | High | 37 | Yes, 65 degrees | >95% ligated and recut | AarI+oligo, Tango2X |
| AscI | Internal | Cutting BioScaffold part into small pieces for removal with spin column | Good to use in combination with low GC content parts | Low | 37 | Yes, 65 (SgsI) | >95%(SgsI), >90%(PalA I) ligated and recut | Tango1X, FastDigest(SgsI); Y(PalA I); NEB 4 |
| BamHI | Other | See BBFRFC15 for use in protein fusion strategies | DNA sequence translates into glycine-serine | Low | 37 | Yes, 80 | >95% ligated and recut | BamHI, G, Tango1X |
| EcoRI | BioBrick | BioBrick | BioBrick | High | 37 | Yes, 65 | > 90% ligated and recut | G, O, W, Y(75%); EcoRI, O, R*, Tango2X, FastDigest; NEB 1,2,3,4 |
| MabI | Internal | Cutting BioScaffold part into small pieces for removal with spin column | Due to recognition sequence, two sites can be used to create a recognition sequence that will not reanneal | Medium | 37 | Yes, 65 | >90% ligated and recut | B(50%), G(75%), O, W, Y(75%) |
| PacI | Internal | Cutting BioScaffold part into small pieces for removal with spin column | Good to use in combination with low GC content parts | Low | 37 | Yes, 65 | 70% ligated, >95% recut | NEB 1 and 4 |
| PpiI | External | Cutting outside BioBrick scars | Good for making protein fusions | High for vectors and protein parts | 30 | Yes, 65 | > 90% ligated and recut | R(100%) or Tango1X,2X(50-100%) |
| PsrI | External | Cutting outside BioBrick scars | Good for making protein fusions | High for vectors and protein parts | 30 | Yes, 65 | 70% ligated, 80% recut (peg improves) | Y |
| PstI | BioBrick | BioBrick | BioBrick | High | 37 | Yes, 80 | >90% ligated and recut | O, Y(50%); B,G,O,T, Tango1X,2X (50-100%), FastDigest;NEB 1(75%),2(75%),3,4(50%) |
| SpeI | BioBrick | BioBrick | BioBrick | High | 37 | No (AhlI), No (BcuI), Yes | >90% ligated and recut (AhlI); >80% ligated, 90% recut (BcuI) | B, G, Y for AhlI; B, G, Tango1X, FastDigest for BcuI; NEB 2 and 4 |
| XbaI | BioBrick | BioBrick | BioBrick | High | 37 | 65 | >90% ligated and recut; >95% | B,G,O,Y; (B,G,Tango2X all 50-100%), Tango2X, FastDigest; NEB 2 and 4 |
| XmaI | Internal | Cutting BioScaffold part into small pieces for removal with spin column | Typically used with AarI parts for historical reasons | Low | 30 | Yes, 65 | >95% ligated, 90% recut | Y |
References
- [http://openwetware.org/wiki/The_BioBricks_Foundation:BBFRFC15 BioBricks Foundation RFC 15] by Julie Norville, Angie Belcher and Tom Knight