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Assembly Standards

What are assembly standards

An assembly standard defines how part samples will be assembled together by the engineer. An assembly standard, like the BioBrick RFC[10], ensures compatibility between parts, allowing part samples to be assembled together creating new longer and more complex parts, while still maintaining the structural elements of the assembly standard. This idempotent assembly means that any newly composed part will adhere to its assembly standard without need for manipulation, and can be used in future assemblies without issue.

How does an assembly standard work

An assembly standard defines assembly through the use of a prefix and suffix (found on plasmid backbones), which flank the beginning and end of a part sample, respectively. The prefix and suffix contain restriction sites, which when used with specific restriction enzymes (cutting) and DNA ligase (connecting), allow part samples to be assembled together forming a new part. This new part sample will maintain the same prefix and suffix as its "parents" and contain a scar, where the cut and re-ligated restriction sites were stitched together. Note: There are also scarless assembly methods, which allow for the assembly of parts without the traditional use of the prefix and suffix restriction sites.

A part is compatible with an assembly standard as long as it meets the requirements defined by said standard. This means that the part will not have any restriction sites that also appear in the prefix and suffix. If there are "illegal" restriction sites in a part this would interfere in its assembly.

Assembly standards in relation too...

  • Part - A part is compatible with an assembly standard, as long as its sequence meets the requirements of said standard; this means that the part does not have any restriction sites that would interfere with the assembly. It is important to remember that a part does not include the prefix and suffix as defined by the assembly standard.
  • Assembly Method is combining two part samples together in series to form a new composite part. Traditional assembly is done through the use of restriction sites (cutting and ligating) as defined by the assembly standard. Assembly methods are facilitated through assembly standards.
  • Plasmid Backbones - A plasmid backbone propagates a sample of a part, located inbetween the prefix and suffix of the plasmid backbone. Therefore the plasmid backbone will define the assembly standard for the part it maintains.


  • You'll notice that many parts on the Registry are BioBrick RFC[10] compatible: BBa_R0051 is a part that is compatible with all five Registry supported assembly standards.
  • If you were to find a physical location for this part (using the Get This Part page) you'll see that all available samples are in the pSB1A2 plasmid backbone.
  • The pSB1A2 plasmid backbone has a prefix and suffix as defined by the BioBrick RFC[10] assembly standard. The available samples of BBa_R0051 are flanked by this prefix and suffix.
  • In order to assemble an available sample of BBa_R0051 to a different part you will need to make sure that a sample of that additional part is on a plasmid backbone that also belongs to the BioBrick RFC[10] assembly standard.

Registry Supported Assembly Standards

The BioBrick standard is the de facto assembly standard for the Registry. All part samples submitted to the Registry must be BioBrick compatible.

Choose your assembly method, support the standard

Choose your assembly method and assemble your parts with a variety of assembly techniques. You can use 3A Assembly, Type IIS, synthesis, Gibson, and more.

Support the standard. Although users can add parts to the Registry that adhere to other assembly standards, BioBrick RFC[10] and Type IIS are the Registry's current de facto standards; all parts on the Registry that will be considered for the iGEM competition (medals, awards, etc.) must be assembly compatible for BioBrick or Type IIS.

In 2019, sample submission is no longer required. However, ensuring your parts are assembly compatible allows the Registry to synthesize your parts in BioBrick and/or Type IIS format. Registry members can easily and reliably use and assemble these parts in the future.

The Registry currently supports the following assembly standards.

RFC 10 The BioBrick™ Standard

The BioBrick RFC[10] is a standard for interchangeable parts based on idempotent assembly. BioBrick RFC[10] is currently the most commonly used assembly standard: the majority of parts in the Registry database are RFC[10] compatible, and the majority of samples in the Registry's Repositories are maintained in RFC[10] plasmid backbones. As such, using RFC[10] ensures a greater diversity when designing your synthetic biology projects.

BioBrick RFC[10] uses an alternate shortened prefix for protein coding regions for optimum spacing between RBS and CDS, creating a 6bp scar instead of the usual 8bp.3

A concern with BioBrick RFC[10] is that it does not facilitate the assembly of protein fusions. If you wish to assemble proteins you will want to use scarless assembly or DNA synthesis.

RFC 12 BioBrick™ BB-2 Standard

The BioBrick BB-2 RFC[12] is a standard for interchangeable parts based on idempotent assembly. RFC[12] was designed to fix the concerns of BioBrick RFC[10], as such it allows for in-frame protein assembly.

Currently BB-2 RFC[12] has not been widely adopted: many parts in the Registry database are compatible but there are few available part samples maintained in BB-2 plasmid backbones. RFC[12] is incompatible with BioBrick RFC[10].

RFC 21 Berkeley Standard

The Berkeley RFC[21] is optimized to enable in-frame assembly of proteins: it is based on idempotent assembly with BamHI and BglII restriction enzymes. RFC[21] is incompatible with BioBrick RFC[10].

Berkeley RFC[21] uses the BglII enzyme, which cannot be heat inactivated, for assembly. Therefore RFC[21] cannot use the 3A Assembly method.

RFC 23 Silver Standard

The Silver RFC[23] is a modification of BioBrick RFC[10] which allows for in-frame assembly of protein domains. RFC[23] shortens the BioBrick prefix and Suffix by 1bp each, allowing for a 6bp scar. RFC[23] has some compatibility with RFC[10].

Although the Silver RFC[23] allows for in-frame protein assembly, the 6bp scar includes an Arg codon which is destabilizing (N-end rule) and will degrade the protein faster.

RFC 25 Freiburg Standard

Freiburg RFC[25] is an extension of the BioBrick RFC[10] and includes two extra restriction sites to allow for in-frame assembly of protein domains. RFC[25] has some compatibility with RFC[10].

Freiburg RFC[25] uses a different prefix (BioBrick RFC[10] CDS prefix) when it is necessary to preserve the N-terminus of a protein.