BglBricks: A flexible standard for biological part assembly

Abstract

BackgroundStandard biological parts, such as BioBricks™ parts, provide the foundation for a new engineering discipline that enables the design and construction of synthetic biological systems with a variety of applications in bioenergy, new materials, therapeutics, and environmental remediation. Although the original BioBricks™ assembly standard has found widespread use, it has several shortcomings that limit its range of potential applications. In particular, the system is not suitable for the construction of protein fusions due to an unfavorable scar sequence that encodes an in-frame stop codon.ResultsHere, we present a similar but new composition standard, called BglBricks, that addresses the scar translation issue associated with the original standard. The new system employs BglII and BamHI restriction enzymes, robust cutters with an extensive history of use, and results in a 6-nucleotide scar sequence encoding glycine-serine, an innocuous peptide linker in most protein fusion applications. We demonstrate the utility of the new standard in three distinct applications, including the construction of constitutively active gene expression devices with a wide range of expression profiles, the construction of chimeric, multi-domain protein fusions, and the targeted integration of functional DNA sequences into specific loci of the E. coli genome.ConclusionsThe BglBrick standard provides a new, more flexible platform from which to generate standard biological parts and automate DNA assembly. Work on BglBrick assembly reactions, as well as on the development of automation and bioinformatics tools, is currently underway. These tools will provide a foundation from which to transform genetic engineering from a technically intensive art into a purely design-based discipline.

Bricks and blueprints: methods and standards for DNA assembly

“…These sequences enable a repeatable, idempotent assembly process: the ligation of two BioBrick parts produces a new, larger BioBrick part with the same physical format. As the BioBrick standard became adopted, various improvements (for example BglBricks) were developed that improved its flexibility and efficiency [14][15][16] .…”

“…These sequences enable a repeatable, idempotent assembly process: the ligation of two BioBrick parts produces a new, larger BioBrick part with the same physical format. As the BioBrick standard became adopted, various improvements (for example BglBricks) were developed that improved its flexibility and efficiency [14][15][16] .Although standards such as BioBricks allow parts to be rationally assembled into desired constructs, it is often quicker to modify existing constructs. New standard plasmid formats have thus been developed to facilitate swapping of parts between constructs.…”

Downward causation by information control in micro-organisms

“…Most of these approaches are based on the present understanding of how natural regulatory pathways work in bacteria [18,19,71]. Because of the high modularity and rather simple regulatory pathways in bacteria (especially when compared with those from eukaryotes), artificial regulatory pathways can be built, and existing functions and molecular parts can be easily exchanged with the new ones (http://partsreg istry.org) [87].…”

The Structure of CcmP, a Tandem Bacterial Microcompartment Domain Protein from the β-Carboxysome, Forms a Subcompartment Within a Microcompartment

“…Mutants-All DNA fragments were amplified from Syn7942 genomic DNA using primers indicated in supplemental Table S4 and cloned using a BglBrick strategy (EcoRI-BglII-partBamHI) (20). The cerulean fluorescent protein was first fused to the C terminus of CcmP before the entire fusion construct was transferred into a BglBrick-modified neutral site I (pAM2991⅐SpR) vector containing an isopropyl 1-thio-␤-D-galactopyranoside-inducible P trc promoter (21) to generate P trc ⅐CcmP-cerulean.…”