The BioBricks Foundation:Standards/Technical/Formats

Biobrick Formats: This working group aims to specify a new BioBrick DNA format.

Aim / Application scenarios for this standard

Formulate a successor to the current BBa physical assembly format of standard biological parts. [add further]

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:

Biobrick alpha format (BBa) (RFC10)

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 Y *

...part B... 3'

description at parts.mit.edu

Advantages

• standard
• well tested and documented
• native protein start codon can be preserved while using RBS parts.
• 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?

BBF RFC 20

This is near identical to RFC 10 but provides a transition plan from moving from using PstI to SbfI which is a 8-bp cutter that is compatible with PstI and includes the PstI in it.

The only modification from the RFC10 standard is that the suffix should be

T ACTAGT A GCGGCCGC CCTGCAGG 3'
   SpeI     NotI    SbfI/PstI

Advantages

• reduced need for mutagenesis
• The set of enzymes EcoRI-HF/XbaI/SpeI/SbfI-HF all have 100% activity in NEBuffer 4 (the standard buffer NEB is pushing for restriction enzymes). PstI isn't good in NEBuffer 4.
• extremely simple transition plan from RFC 10 parts. Any RFC 10 part can be made into a RFC 20 part either by simply moving the part into a different plasmid or during a normal assembly with a RFC 20 suffix part.

Disadvantages

• All of the disadvantages of RFC 10
• May be confusing to mix RFC 10/RFC 20 parts

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 -- no new enzymes
• also protein parts can, theoretically, be fused N-terminally to to BBa protein parts, as long as the frameshift is corrected by an adapter part
• used by several iGem teams

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 (but equivalent to BBa coding part could be constructed)

Fusion parts (Freiburg iGem2007 team)

The Freiburg iGem2007 team proposed an extension of BBa, which would enable protein fusions but alleviate the disadvantages of the Biofusion format. Two restriction sites are added within the standard BBA sites. These additional sites provide compatible ends, can be employed using the same cloning strategy as for the standard restriction sites, and code for amino acids suited for linkers:

Prefix		Suffix
5' GAATTC GCGGCCGC T TCTAGA TG GCCGGC EcoRI NotI XbaI Met NgoMIV (=NgoMI)	...part...	ACCGGT TAAT ACTAGT A GCGGCCG CTGCAG 3' AgeI * SpeI NotI PstI
"N-part" prefix
5' GAATTC GCGGCCGC T TCTAG EcoRI NotI XbaI	ATG.part

Fusing two parts based on the new, "fusion"-restriction sites leaves the following scar:

5' ...part A...

ACCGGC T G

...part B... 3'

description by Freiburg iGem team

For cases where the native ATG needs to be conserved, the Freiburg team introduces an "N-part" which has the classic BBa coding part prefix and the Fusion part suffix. N-parts would need to be cut with XbaI in place of NgoMIV.

NgoMIV has recently been renamed to NgoMI, this enzyme has the isoschizomers MroNI and NgoAIV and the blunt end nesoschizomers NaeI and PdiI. AgeI has the isoschizomers PinAI, CspAI, AsiI, AsrGI, and BshTI.

Advantages

• in-frame fusion of protein parts
• benign protein scar
• N-end rule safe (long protein half-life)
• provision for preserving native N-terminal while using RBS parts
• both new enzymes can be heat-inactivated
• stand-alone protein expression (start + stop in prefix / suffix)
• full BBa compatibility -- functionally & compositionally equivalent to BBa protein coding part
• blunt-cutting isochizomer of NgoMIV (NaeI) -- possibility of directional cloning with two inner restriction sites enables part transfer between different formats and other potentially interesting transfer reactions.

Disadvantages

• N-parts are assembled with a different enzyme combination.
• not compatible to BioFusion protein parts (frame shift + stop codon), but see below!

Fusion / BioFusion format conversion strategies Until recently, the Silver lab BioFusion format was the only well-described way to compose fusion proteins from (unofficial) BioBricks. Due to the frameshift, the Fusion Part format cannot be made compatible to both BBa and BioFusion at the same time. Since I (Raik) am collaborating with BioFusion users, I am constructing a conversion vector that introduces a prefix and suffix that are compatible to both BioFusion and Fusion Parts. I would not consider this a good candidate for a real format though. Fusion -> BioFusion conversion vectors Construction vectors with a modified Freiburg prefix / suffix could bring a protein part in frame with BioFusion parts and remove the STOP between the AgeI and SpeI site. Restriction / ligation with AgeI + NaeI can (theoretically) transfer Fusion parts into this conversion vector which can then be used for normal BioFusion cloning (at the cost of adding a T G before and after the part). NaeI is an isoschizomer to NgoMIV but generates blunt ends which should allow for a directional transfer. 5' GAATTC GCGGCCGC T TCTAGA GCCGGC EcoRI NotI XbaI NgoMIV ...part... ACCGGT ACTAGT A GCGGCCG CTGCAG 3' AgeI SpeI NotI PstI BioFusion --> Fusion part conversion None really. Introducing the modified flanks by PCR seems the only way.

The Berkeley (BBb) Format (now called BglBricks)

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:

Prefix		Suffix
5' GAATTC atg AGATCT EcoRI BglII	...part...	GGATCC taa CTCGAG 3' BamHI * XhoI

Fusing two parts leaves the following scar:

5' ...part A...

GGATCT G S

...part B... 3'

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

Advantages

• in-frame fusion of protein parts
• benign protein scar
• enzymes selected for efficient cutting

Disadvantages

• BglII cannot be heat-inactivated -- current 3A standard assembly won't work
• incompatible to BBa format
• incompatible to BioFusion format

Tom Knight's BBb proposal

Tom Knight has recently proposed to replace BBa by the following format:

Prefix		Suffix
5' GAATTC...ACTAGT EcoRI SpeI	...part...	GCTAGC...CTCGAG 3' NheI PstI

Fusing two parts would then leave the following scar:

5' ...part A...

GCTAGT A S

...part B... 3'

Advantages

• in-frame fusion of protein parts
• benign protein scar
• N-end rule save
• introduction of only one new enzyme
• NheI is comparatively rare in the E. coli genome -- less background fragments from genomic DNA
• NheI can be heat-inactivated

Disadvantages

• incompatible to BBa format
• incompatible to BioFusion format

Comparison of different format proposals

	BBa	Silver	Freiburg	Berkeley	Knight
protein issues
(1) protein fusion?	no	yes	yes	yes	yes
(2) good protein scar?	n/a	no	yes	yes	yes
(3) N-end rule save?	n/a	no	yes	yes	yes
(4a) native N' after RBS part?	coding part	pRBS?	N-part	pRBS?	pRBS?
(4b) native N' after Kozak part?	no	no	no	no	no
BBa compatibility
(5) same ass. enzymes?	yes	yes	no	no	no
(6) restriction-compatible?	yes	yes	yes	no	no
(7) ..side effects?	none	some	none	n/a	n/a
(8) directional transfer with inner restr. sites?	no	no	sticky/blunt ligation	no	no
standard assembly issues
(9) heat inactivation?	yes	yes	yes	no	yes
(10)inner restr. site occurrence in E. coli	240	240	2100	1200	500
(11) enzyme efficiency?	ok	ok	?	good	?
other issues
(12) current adoption?	wide	some	some	some	none

Explanations:

_{(2) good meaning small and biochemically nonperturbing}

(3) Some N-terminal amino acids are strong destabilization signals in both pro- and eucaryotes. The assembly scar turns into the protein N-terminal if a RBS+start part is coupled with the protein part -- the scar should hence not code for destabilizing residues.

(4a) Some protein parts, in particular signaling peptides, critically depend on preserving their sequence at the N-terminal. That means any additional scar residues between a "start" part and the old N-terminal would disrupt function. Procaryotic RBS tolerate a variable 6bp spacer between RBS and AUG and special parts-layout can shift the assembly scar into this spacer. BBa and Freiburg define a specialized sub-format where the ATG is shifted into the part and partly overlaps with the end of the scar. pRBS?: These formats make no provision for a native ATG but still use a 6 bp scar and one could thus design special RBS parts that do *not* include the ATG. see: RBS, Kozak explanation

(4b) All bets are, apparently, off for eucaryotic proteins where the Kozak reaches 4 bp (ATG G) into the reading frame. Only a scar-less assembly method would allow us to freely combine special Kozak parts (lacking ATGG) with special N-term protein parts (starting with ATG). Kozak and N-terminal signaling peptides are thus not decomposable with any of the current schemes.

(5) Uses same restriction enzyme combination as the BBa assembly.

(6) The format allows to couple parts with "old" BBa parts using the BBa assembly.

(7) Side effects mean that parts in the new format do not behave exactly like BBa parts after assembly with "old" BBa parts.

(8) Can the part be directionally transferred into other vectors that introduce different flanking sequences (more or less) directly right and left of it? This may be critical for offsite-cutter (IIS) based or other future assembly schemes.

(9) Can all restriction enzymes used be heat-inactivated?

(10) Approximate combined occurrence of the two inner restriction sites in E. coli.

entirely 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 may not behave as ideally as assumed (?) (5) Several of the proposed "operations" involve two sequential restriction/ligation/transformation cycles which, in practice, 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.

Physical Assembly Standards, Naming Revision Proposal

Randy Rettberg has noted that the "alpha" in "BBa" (e.g., as found with many or all of the parts in his Registry) does not refer to a particular assembly standard. Rather, the "alpha" refers to an early collection of parts, which are likely not yet very good. In other words, "BBb" would refer to a higher quality collection of parts (e.g., better support for functional composition). This leads to two items for discussion.

1. Should we rename all physical composition standards along the lines of BioBrick Assembly Standard #1, #2, #3, et cetera?
2. What defines whether or not a part is "alpha," "beta," or something of still higher quality?