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 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.}

description at parts.mit.edu

Advantages

• de-facto standard
• well tested and documented
• native protein start codon can be preserved

Disadvantages

• no protein fusions (frame shift, stop codon)
• a single mutation (at the fused region) can upset the setup?

"2.0" Biofusion (Silver lab)

The Silver lab modified the classic format to allow for protein fusions:

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 1.0 coding)

"3.0" (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:

description by Freiburg iGem team

Advantages

• in-frame fusion of protein parts
• less intrusive protein scar
• N-end rule safe (longer protein half-life)
• stand-alone protein expression (start + stop in prefix / suffix)
• compatible to 1.0 and 2.0 non-coding parts

Disadvantages

• stand-alone protein expression (start + stop in prefix / suffix)
• not compatible to Biofusion protein parts (frame shift + stop codon)
• not possible to preserve native protein start (as in 1.0 coding)

"2.0-3.0" (CRG proposal)

Biobrick users at the CRG propose a modification to the Freiburg format which would make 3.0 and 2.0 biobricks compatible with each other and allow 3.0 biobricks to be fused N-terminally to 1.0 protein coding parts. The modification sacrifices the possibility to have a single coding biobrick ready for expression (start and stop in the prefix and suffix are deleted). This may also be considered am advantage though, as it somewhat reduces the risk of uncontrolled expression leading to toxicity.

Advantages

• in-frame fusion of protein parts
• less intrusive protein scar
• N-end rule safe (longer protein half-life)
• full backwards-compatibility (coding and non-coding 1.0 and 2.0 parts)

Disadvantages

• no stand-alone protein expression
• not tested

different strategies

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