The BioBricks Foundation:Standards/Technical/Formats
Biobrick Formats: This working group aims to specify Biobrick DNA formats.
Aim / Application scenarios for this standard
[ add ]
Overview over existing and proposed Biobrick formats
All biobrick formats proposed so far follow the same basic scheme where restriction and ligation of two biobricks forms a new biobrick.
classic 1.0 Biobrick format
This is the classic Biobrick format used by most iGem teams and most biobricks in the MIT registry.
Note, for coding parts, the prefix is shortened so that the ATG is fully part of the biobrick sequence.
Advantages
- de-facto standard
- well tested and documented
- native protein start codon can be preserved
- large and still growing set of parts
Disadvantages
- no protein fusions (frame shift, stop codon)
- a single mutation (at the fused region) can upset the setup?
Biofusion (Silver lab)
The Silver lab modified the classic 1.0 format to allow for protein fusions:
Advantages
- in-frame fusion of protein parts
- restriction-compatible to 1.0 parts
- protein parts can, theoretically, be fused N-terminally to to 1.0 protein parts, as long as the frameshift is corrected by an adapter part
Disadvantages
- Arg in scar can be problematic
- N-terminal Thr-Arg = destabilization signal (N-end rule)
- Dam methylation blocks cloning when prefix is followed by "TC"
- unexpected side-effects for users not aware of the shortened prefix/suffix
- non-coding parts may be not functionally compatible due to the changed bp distance
- frameshift with respect to what is expected from protein coding 1.0 parts
- not possible to preserve native protein start (as in 1.0 coding)
3.0 Expression parts (Freiburg iGem team)
The Freiburg 2007iGem team proposed a more radical modification or rather extension of 1.0, which would enable protein fusions but alleviate the disadvantages of the Biofusion format:
Typo! the prefix GGCGCC site is for NgoMIV whereas AgeI is cutting the suffix ACCGGT!
description by Freiburg iGem team
For cases where the native ATG is to be conserved, the Freiburg team allows an "N-part" which has the 1.0 coding part prefix and the Expression part suffix. N-parts would need to be cut with XbaI in place of NgoMIV.
Advantages
- in-frame fusion of protein parts
- benign protein scar
- N-end rule safe (long protein half-life)
- stand-alone protein expression (start + stop in prefix / suffix)
- full 1.0 compatibility -- functionally & compositionally equivalent to 1.0 coding part
Disadvantages
- stand-alone protein expression (start + stop in prefix / suffix) -- toxicity?
- not compatible to Biofusion protein parts (frame shift + stop codon)
- cannot preserve native protein start AND preserve 1.0 inter-part distance at the same time
3.0 -> Biofusion adapter parts
- C-terminal adapter: a part ending in an incomplete 2-bp codon
...would correct the frameshift to Biofusion and override the STOP codon. Biofusion parts could then be appended to the adapter using 1.0 restriction. The resulting scar would be ugly though: P V N (T R)
- N-terminal adapter: a part beginning with a single stray nucleotide
...would allow to allow to couple Biofusion parts in front of the adapter. The scar would be again ugly: (T R) W P R (regardless of the stray nucleotide).
3.0 / Biofusion compatibility format
A modification to the Freiburg format which would make 3.0 and Biofusion biobricks compatible with each other but largely break the compatibility to 1.0. The main use case for this format would be as a construction intermediate before a 3.0 part is mixed into Biofusion parts. Restriction with AgeI + NaeI can (theoretically) transfer parts between 3.0 and this format. NaeI is an isoschizomer to NgoMIV but generates blunt ends which should allow for a directional transfer.
Typo! the prefix GGCGCC site is for NgoMIV whereas AgeI is cutting the suffix ACCGGT!
Advantages
- in-frame fusion of protein parts
- benign protein scar
- N-end rule safe (longer protein half-life)
- Biofusion compatible (with default Biofusion scar and without adapters)
- 3.0 compatible
Disadvantages
- unexpected side-effects for 1.0 users not aware of shortened prefix/suffix:
- very different separation if combined with 1.0 upstream and downstream parts
- frameshift with respect to what is expected from protein coding 1.0 parts
- no self-sustained expression (start + stop) as expected from 1.0 protein coding parts
- not possible to preserve native protein start (as in 1.0 coding)
- not tested
different strategies
- IIS restriction strategy from the UCSF iGem2007 team could probably be extended into a more general multi-ligation Biobrick system:
The BBb Format
BBb is used by several researchers at UC Berkeley and is based on idempotent assembly with BamHI and BglII restriction enzymes. In a nutshell, most plasmids look like this:
GAATTCatgAGATCT-part-GGATCCtaaCTCGAG
and BBb scars are "GGATCT" encoding gly-ser when translated in frame. Note, however, that BBb is intended as a minimal physical assembly standard, and only those features needed for interconversion of BBb plasmids are formally defined. Therefore, "atg" and "taa" spacers are not core definitions of the standard.
Formal Definition:
- A BBb part is a DNA sequence flanked on the 5' end by "GATCT" and on the 3' end by "G" lacking BglII, BamHI, EcoRI, and XhoI restriction sites
- A BBb vector is a DNA sequence flanked on its 5' end by "GATCC" and on its 3' end by "A"
- A BBb entry vector has a unique EcoRI site, no BamHI or BglII restriction sites, and at most one XhoI site 5' to the EcoRI site
- A BBb plasmid is represented as <vector_name>-<part_name> and has the sequence obtained by concatenating the vector and part sequences
- Further definition constraints are "sub-standards" of the BBb format
