Improved BioBrick Standard

For a multitude reasons we would like to work with the upgrade of 2007 Freiburg iGEM proposed standard (Assembly standard 25). The upgrade includes altered multiple-cloning site which enables friendly scar after part ligation and simple extension of linker between parts.

Advantages:

• in-frame fusion of protein parts
• benign protein scar/scars
• preserving standard restriction sites of prefix and suffix
• four new restriction sites are added in multiple-cloning site
• heat inactivation??
• no Dam methylation problem for XbaI
• stand-alone protein expression
• full BBb compatibility and
• blunt-cutting isochizomer of NgoMIV (NaeI) and XmaI (SmaI) possibility of directional cloning with two

restriction enzymes enables part transfer between different formats and other potentially interesting transfer reactions

Disadvantages:

• unexpected site effects for users not aware of different prefix/suffix
• N-parts could be assembled with different enzyme combination
• not compatible to BioFusion format (frame shift; stop codon)
• not compatible to BBb format (Berkeley format)
• additional limitations on the nucleotide sequence to avoid additional restriction sites

Here we describe sequence properties of modified vector BioBrick-NIC-II. All sequences defined herein are specified in the 5' to 3' direction.

A: BioBrick-NIC-II originates from standard Biobrick vector

The difference is in multiple cloning sites.

The main reasons for changing multiple cloning sites are:

1. Defined position of promoter and terminator from coding sequence coded with part or parts, which ensures efficient transcription and translation of protein; and
2. Additional sequences linked to parts, which could be exploited when linker is needed between combined parts.

Although most promoters and RBS and tags and terminators are currently specified as separate parts in the Registry, we will use a new design in which these elements are included within the vector, as we plan to prepare and isolate many proteins and this design will also decrease the number of required cloning steps and will be suesull for others desiring to prepare recombinant proteins in E.coli. We expect the new design will reduce the likelihood of unexpected functional composition problems between promoter / terminator and coding sequence.

The BioBrick-NIC-II vector is especially appropriate for cloning and combining parts coding short peptides.

The current assembly process requires certain sequence properties for the part and the surrounding DNA.

prefix: 5' gaattc(EcoRI) gcggccgc(NotI) promoter (invariant region) tctaga(XbaI) gccggc(NgoMIV) accggt(AgeI)

suffix: 5' cccggg(XmaI) tccgga(BspEI) terminator (invariant region) gcggccg(NotI) ctgcag(PstI)

B: Multiple-cloning site