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 [1] 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: as a member you may 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 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 solicted 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 [[2]] (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 contemplete) so that others in the community do not duplicate your efforts. As the community grows we will create a mailing list.


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


Currently, BioScaffold parts and their status must be manually added to the following table. The restriction enzymes that should be removed to make vectors compatible with all the current versions of BioScaffold parts are PpiI, PsrI, AarI, and MabI. XmaI is being considered, but is not currently considered one of the essential BioScaffold enzymes. AscI and PacI are sometimes used instead of MabI because their restriction sites make them othogonal to many parts based on GC content, but are not currently considered essential for removal.
-?- 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 (use J70040 instead, currently) AarI XmaI
β Part:BBa_J70036 (use J70050 and J70052 instead, currently) AarI XmaI
β Part:BBa_J70040 A BioScaffold Part (for Library Vector Preparation) AarI MabI
β/γ Part:BBa_J70050 A BioScaffold Part (for Library Insert Preparation) AarI AarI
β/γ Part:BBa_J70052 A BioScaffold Part (for Library Insert Preparation) AarI AarI


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


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 [3] PpiI(2, V) AarI, AscI, BamHI, MabI, PacI, PsrI, XmaI No
pSB1AC3, high copy [4] PpiI(2, H) AarI, AscI, BamHI, MabI, PacI, PsrI, XmaI Yes
pSB1AK3, high copy [5] PpiI(2, H) AarI, AscI, BamHI, MabI, PacI, PsrI, XmaI Yes
pSB1AT3, high copy [6] PpiI(2, H), BamHI(?,M) AarI, AscI, MabI, PacI, PsrI, XmaI Yes
pSB1A7, transcriptionally insulated high copy [7] 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 [8] BamHI(?, L) AarI, AscI, MabI, PacI, PpiI, PsrI, XmaI Yes
pSB3C5, low to medium copy [9] 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 [10] PpiI(1, M) and PsrI(1, M) AarI, AscI, BamHI, MabI, PacI, XmaI Yes
pSB4A3, discontinued [11] PpiI(1, V) AarI, AscI, BamHI, MabI, PacI, PsrI, XmaI No
pSB3K5, low to medium copy standard vector [12] PpiI(1, M) AarI, AscI, BamHI, MabI, PacI, PsrI, XmaI Yes
pSB4C5, low copy standard vector [13] None AarI, AscI, BamHI, MabI, PacI, PpiI, PsrI, XmaI Yes
pSB4K5, low copy standard vector [14] None AarI, AscI, BamHI, MabI, PpiI, PsrI, XmaI Yes
pSB4A5, low copy standard vector [15] PpiI(1, H) AarI, AscI, BamHI, MabI, PacI, PsrI, XmaI Yes
pSB4T5, low copy standard vector [16] PsrI(1, H) AarI, AscI, BamHI, MabI, PacI, PpiI, XmaI Yes