The BioBricks Foundation:Standards/Technical/Formats

Biobrick Formats: This working group aims to specify Biobrick DNA formats.

Aim / Application scenarios for this standard

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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 Biobrick format (BBa)

This is the format used by most iGem teams and most BioBricks in the MIT registry.

prefix		suffix
5' GAATTC GCGGCCGC T TCTAGA G EcoRI NotI XbaI	...part...	T ACTAGT A GCGGCCG CTGCAG 3' SpeI NotI PstI
Protein coding prefix
5' GAATTC GCGGCCGC T TCTAG EcoRI NotI XbaI	ATG.part

Fusing two parts leaves the following scar:

5' ...part A...

TACTAGAG * *

...part B... 3'

description at parts.mit.edu

Advantages

• 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:

Prefix		Suffix
5' GAATTC GCGGCCGC T TCTAGA EcoRI NotI XbaI	...part...	ACTAGT A GCGGCCG CTGCAG 3' SpeI NotI PstI

Fusing two parts now leaves the following scar:

5' ...part A...

ACTAGA T R

...part B... 3'

description by Silver lab

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! The sequence is correct but the Enzyme labelling is wrong.}

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
• blunt-cutting isochizomer of NgoMIV (NaeI) -- possibility of directional cloning with two inner restriction sites enables part transfer between different formats and many more potentially interesting transfer reactions.

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 (unless using special N-part)

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! The sequence is correct but the Enzyme labelling is wrong.}

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

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

different strategies

IIS restriction and multi-fragment ligation

The IIS restriction strategy from the UCSF iGem2007 team could probably be extended into a more general multi-ligation Biobrick system: UCSF 2007 cloning strategy

BioBrick ++

... was an early (2004) proposal for a more versatile BioBrick format, which somehow didn't catch on. BioBrick ++ is based on a sophisticated combination of IIS (offsite cutters) and nicking restriction enzymes, and was intended to allow both seamless and normal BioBrick assembly, flipping of BioBricks and other operations. There are some disadvantages though [Raik's opinion, add your own view]:

(1) the large combination of restriction sites makes the system not quite easy to understand. (2) ++ was designed without keeping protein fusions in mind -- the proposed standard assembly would again introduce a frameshift and a stop codon, although the more sophisticated blunt assembly would of course work for protein fragments. (3) the different assembly methods produce different frames. (4) Some of the proposed enzymes or ligation schemes are rather exotic and may not behave as ideally as assumed (?) (5) Several of the proposed "operations" involve two sequential restriction/ligation/transformation cycles which, in practise, may amount to more work then a normal single step conversion by PCR.

Nevertheless, BioBrick ++ describes, at least, two core innovations that may be very helpful for a second (or third?) generation BioBrick format:

1. IIS-restriction (offsite cutting) in prefix and suffix uncouples the cohesive ends from the enzyme recognition sites -- overhangs can therefore end directly at the part boundary (allowing for blunt ligation strategies and parts "upgrade")
2. Construction plasmids can be created with any overhang by inside-out IIS restriction or with nicking enzymes.