Help:Assembly standard 15

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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 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

  1. BioBricks Foundation RFC 15 by Julie Norville, Angie Belcher and Tom Knight