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: 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 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, 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 [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