The BioBricks Foundation:BBFRFC22

BBFRFC22 BBΩ-- An Extended BioBricks Assembly Standard that Utilizes Hierarchical and Orthogonal Manipulation of Parts to Address Limitations in the Original BioBricks Assembly Standard Julie Norville, Angela Belcher, and Tom Knight Date: (Draft standard-April 16, 2009)

1 Purpose

The original BioBricks standard (BBF RFC 10) is limited, as it primarily allows idempotent transformations of parts (idempotent assembly). There are many types of DNA manipulation that it does not support. BBF RFC 22 describes a new and extended version of BBF RFC 10 that continues to allow idempotent assembly but also allows hierarchical and orthogonal assembly in order to give parts and device designers greater freedom. BBF RFC 22 lays the framework so that BBF RFC 22 may itself be extended in a non-destructive fashion.

Related Requests for Comments: BBF RFC 8, BBF RFC 9, BBF RFC 11, BBF RFC 12, BBF RFC 14, BBF RFC 15, BBF RFC 21, BBF RFC 23, BBF RFC 25, BBF RFC 26, BBF RFC 27 (?), BBF RFC 28

2 Introduction

BBF RFC 10 or standard assembly frees users from many limitations of standard cloning, yet it also constrains parts and device designers from building many systems that they would desire using parts composition. Historically, the inability to create protein fusions was the first practical limitation of BBF RFC 10 to be identified and addressed by the community. Beginning with work by Silver, a variety of new standards were created to address the inability to two compose two protein parts in a sensible fashion (BBF RFC 3, 12, 21, 23, 25.) Though Silver's standard (BBF RFC 23) was compatible with BBF RFC 10 for applications that use non-methylated DNA, Anderson's standard (BBF RFC 21) made a compelling case to change the restriction enzymes used to implement this new variant of idempotent assembly. The new standards that followed (BBF RFC 3, 12, 14) also made changes in the the enzymes used in idempotent assembly or added additional adaptor enzymes.

The protein fusion problem allows one to gain a sense of the costs of standard switching, simply to change the enzymes used in idempotent assembly. There are dollar, time, and community conversion costs in changing "part-ends" and removing restriction enzymes such that new a full or partially idempotent assembly standard will be supported. Though the costs may be justified if conversion only needed to happen once or a few times, it is inevitable that protein fusions are not the only problem that will need to be addressed in synthetic biology and it is not yet clear that allowing the creation of protein fusions should be supported if doing so excludes the ability to execute other valuable DNA manipulations. This leaves the field in a dilemma, because many compelling ideas are emerging yet it is hard to incorporate one idea without excluding others, especially if the physical implementation of the idea requires exclusive conversion to a new idempotent assembly standard. For instance in BBF RFC 20, Che suggests an idempotent assembly standard that could sharply cut down on the number of restriction sites that need to be eliminated in order to domesticate a part by converting it into a BioBrick. Unfortunately, accepting BBF RFC 20 will require conversion of the over 3000 parts in the Registry of Standard Biological Parts to this new idempotent assembly standard. Anderson has done early work in bringing the ability to construct libraries to the Synthetic Biology community (which has the potential to emerge as a foundational technology that is critical in tuning parts to create functional devices,) however, idempotent assembly as described in BBF RFC 10 is not currently conducive to library fabrication.

3 Proposal

Assembly standards describe how part composition occurs. To date proposed standards primarily describe idempotent or extended-idempotent (with adaptors) frameworks. However, the conflicts emerging in this standards space indicate that idempotent assembly, though necessary for (and REQUIRED by the Synthetic Biology community, is not sufficient. A more flexible means to compose parts is REQUIRED. Since idempotent assembly has many advantages, we propose that it SHOULD be retained by the community. We propose that hierarchical, orthogonal, and additional assembly standards be implemented that free parts and device designers from the constraints of idempotent assembly, yet do not interfere with the idempotent form of assemblies.

We RECOMMEND that the currently accepted standard BBF RFC 10 be retained for the time being. Retaining the ability to compose parts using the form of idempotent assembly described in BBF RFC 10 is currently desirable due to the large collection of BBC RFC 10 compatible parts in the Registry. Currently, both the needs of the community and ideas for assembly are continuing to evolve.

We RECOMMEND that the community begins to explore new ideas for hierarchical and orthogonal assembly standards. In order to be considered, proposed hierarchical and orthogonal assembly standards SHOULD support at least one form of idempotent assembly. In BBFRFC 22 we provided examples of hierarchical and orthogonal assembly and the parts that we suggest that they be implemented with do not interfere with the original BBF RFC 10 idempotent assembly standard. In order to be compatible with the new BBFRFC 22 standard, new proposed forms of hierarchical or orthogonal assembly SHOULD not interfere with the original BBFRFC 10 standard, though at present the proposed hierarchical and orthogonal assembly standards MAY interfere with each other. We expect that certain classes of hierarchical and orthogonal assembly standards will become dominant.

We PROPOSE that hierarchical and orthogonal assembly standards MAY be created using the new class of BioBrick parts described in BBFRFC 15. Hierarchical and orthogonal assembly MAY be implemented in many different fashions and we invite the community to extend BBFRFC 22 by suggesting new implementations for hierarchical and orthogonal assembly that MAY or MAY NOT use the parts described in BBFRFC 15.

Idempotent, hierarchical, and orthogonal assembly MAY be implemented as follows:

Idempotent assembly:

<Part A>+<Part B> yields <Part A | Part B>, where <> indicates the idempotent ends on an assembly that retain their structure (in the the preferred implementation the ends specified in BBFRFC 10 SHOULD be utilized.)

Hierarchical Assembly:

<Part A|Hierarchical Part(such as a BioScaffold Part)|Part B> + <Part C> + <Part D> + <Part E> + <Part F> MAY yeild after some manipulation (and assuming good design and efficient assembly) <Part A|Part C|Part B> and <Part A|Part D|Part B> and <Part A|Part E|Part B> and <Part A|Part F|Part> By using the BioScaffold parts described in BBFRFC 15, one MAY perform hierarcical assembly and even use it for applications like protein fusions, library fabrication, or inserting a non-domesticated (i.e., non-BioBricked parts) or shortened oligonucleotide part.

Orthogonal Assembly

{<Part A>}+[<Part B>] yields <Part A | Part B>, where <> indicate the standard idempotent ends even though the assembly after non-standard manipulations that were orthogonal to the selected idempotent assembly standard (in this case, BBFRFC should be used.) Orthogonal Assembly is intended to give the parts and device designers more freedom. By using the BioScaffold parts described in BBFRFC 15, one may perform orthogonal assembly, which may be especially useful in manipulating non-domesticated (i.e., non-BioBricked parts.) One even has the potential to standardize ideas from Peisojovich in BBFRFC 28 to create rapid parts fabrication protocols.

(Rename) New forms of assembly SHOULD be proposed as follows:

(Rename) New Forms of Assembly

New forms of BBFRFC 22(X) compatible assembly methods SHOULD described as such provided that they do not interfere with idempotent assembly standard BBFRFC X cannot be completely described by hierarchical or orthogonal assembly.

Relationships of the different types of standards

Idempotent assembly will be the primary assembly standard for a class of parts. Usually idempotent assembly will be used first to create the part assembly or device framework. Hierarchical and orthogonal assembly are secondary assembly standards for a class of parts. Secondary assembly strategies will typically be used less frequently than primary assembly strategies. Hierarchical assembly is typically used to tune or make small manipulations in an assembly. Orthogonal assembly is typically used to create niche parts for prototypes.