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 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:

description by Freiburg iGem team

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.

Advantages

• in-frame fusion of protein parts
• benign protein scar
• N-end rule safe (longer protein half-life)
• Biofusion compatible
• 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
• not possible to preserve native protein start (as 1.0 coding)
• not tested

different strategies

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