BglBricks: A flexible standard for biological part assembly

Abstract

Background: Standard 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.

Results: Here, 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.

Conclusions: The 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.

Figure 1

Standard assembly of BglBrick parts. Unique BglII (in red) and BamHI (in blue) restriction sites flank BglBrick basic parts on their 5' and 3' ends, respectively. EcoRI and XhoI restriction sites employed in various protocols for part assembly are also shown. Cleavage of each DNA with the appropriate enzyme (color-coded arrowheads) generates compatible cohesive ends. These can be connected head-to-tail by ligation (black arrow) to generate composite parts separated by a 6 nucleotide scar sequence (ggatct). When translated in frame, the scar sequence between parts encodes glycine-serine, a peptide linker innocuous for most protein fusion applications.

Figure 2

Assembly of lacZ expression devices. (A) Standard assembly of BglBrick basic parts were used to generate a series of constitutive lacZ expression devices. Each composite part consisted of a different ribosome binding site part located between promoter and lacZ coding sequence parts. (B) Sequence alignment of RBS variants. Asterisks indicate sequence identity. (C) The β-galactosidase activity of each device (labeled for RBS parts J61140-J61146), along with a negative control lacking a device (DH10B cells alone), is shown. For each, the average of 5 individual replicates is shown. A wide range of activities were observed, indicating the ability to tune protein expression levels using BglBrick RBS part libraries.

Figure 3

Assembly of multi-domain fusion protein devices. (A) Basic parts containing protein-protein interaction ligands were built using three independent, degenerate sequences encoding an N-terminal glycine-serine linker and an SH3 interaction peptide. Composite parts were created by standard assembly of these basic parts. (B) Experimental design for testing interactions between bait and prey parts. The number of prey molecules pulled-down by bait should be dependent on the number of SH3 peptide ligands. (C) A GST-pulldown experiment was conducted and the proteins separated via SDS-PAGE and then imaged by Coomassie staining. No visible interaction was observed when either the bait lacked any SH3 peptide ligand or the prey lacked an SH3 domain. Bait containing composite part J62008 with four SH3 peptide ligands, despite visible proteolytic degradation, was sufficient to pull down increased amounts of prey compared to bait containing the single SH3 ligand part J62002.

Figure 4

Targeted integration of BglBrick parts into the E. coli genome. (A) A variety of basic parts were used to create two methyltransferase expression devices targeting BamHI and BglII restriction sites (parts J72007 and J72013, respectively). Each device was recombined into the genome of strain MC1061 by Φ80 att site integration, resulting in BamHI- and BglII-methylating strains (parts J72015 and J72014, respectively). (B) Sample experimental design for genomic integration of CRIM plasmids. Circuit components are color-coded and graphically represented relative to (A) and the top of (B) for easy identification. (1) Host strain MC1061 with unmodified genomic Φ80 att sites is transformed with temperature sensitive helper plasmid pInt80-649 (part J2008) and selected on ampicillin plates. (2) Cells are re-transformed with CRIM plasmid (part J72007), which replicates as a high-copy R6K plasmid employing the pir gene provided by the helper plasmid. (3) The CRIM plasmid inserts into the genome by recombination with the Φ80 att site employing the int gene from the helper plasmid. (4) Helper plasmid is cured by growth at 43°C. (5) Helper plasmid pCP20 encoding Flp recombinase is introduced by transformation and the R6K origin and chloramphenicol genes are excised from the genome by recombination of FRT sites. (6) Helper plasmid is cured by growth at 43°C, resulting in the final product containing a genomically-integrated BglBrick part and a single FRT site. (C) Restriction mapping of plasmid J61148-J72011 isolated from BglII- and BamHI- methylating strains (parts J72015 and J72014, respectively) or DH10B (control) confirms the appropriate protection.