BioScaffold Parts
| BioScaffold parts are BioBrick parts that contain restriction enzyme recognition sites that enable their full or partial removal from a BioBrick assembly. |
The document BBFRFC15 [http://openwetware.org/wiki/The_BioBricks_Foundation:BBFRFC15] describes some potential parts, uses, and format for BioScaffold parts. BioScaffold parts help extend BioBrick standard assembly to many new applications and can overcome many of its limitations (such as those regarding protein fusions, library fabrication, and part domestication.) You may wish to become a part of the BioScaffold community: to do so you create and test already designed BioScaffold parts, 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 csail 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 solicted as a co-author on these documents. You may also wish to describe your intent on the BioScaffold Part User Group wiki http://openwetware.org/wiki/BioScaffold_Parts_Users_Group (especially if you wish to convert plasmids or other important parts to a BioScaffold part compatible format) so that others in the community do not duplicate your efforts. As the community grows we will create a mailing list.
| 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) |
| 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 |
| -?- | Class | Name | Description | External Enzymes | Internal Enzymes |
|---|---|---|---|---|---|
| A | α | Part:BBa_J70010 | A BioScaffold Part (for offset see Part Design page) | PpiI | MabI, PacI |
| A | α | Part:BBa_J70012 | A BioScaffold Part (Protein Head Remover) | PsrI | MabI, AscI |
| α | Part:BBa_J70030 | A BioScaffold Part (Protein Tail Remover/Protein Fusions) | PpiI | MabI, PacI | |
| α | Part:BBa_J70032 | A Composite BioScaffold Part (Protein Fusions) | PpiI, PsrI | MabI, (AscI and PacI) | |
| β | Part:BBa_J70034 | A BioScaffold Part (for Library Vector Preparation) | AarI | XmaI | |
| β | Part:BBa_J70036 | A BioScaffold Part (for Library Insert Preparation) | AarI | XmaI |
Plasmid Compatibility with BioScaffold Part Internal and External Enzymes
| 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 |
| 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 |