BglBrick vectors and datasheets: A synthetic biology platform for gene expression

Abstract

Background: As engineered biological systems become more complex, it is increasingly common to express multiple operons from different plasmids and inducible expression systems within a single host cell. Optimizing such systems often requires screening combinations of origins of replication, expression systems, and antibiotic markers. This procedure is hampered by a lack of quantitative data on how these components behave when more than one origin of replication or expression system are used simultaneously. Additionally, this process can be time consuming as it often requires the creation of new vectors or cloning into existing but disparate vectors.

Results: Here, we report the development and characterization of a library of expression vectors compatible with the BglBrick standard (BBF RFC 21). We have designed and constructed 96 BglBrick-compatible plasmids with a combination of replication origins, antibiotic resistance genes, and inducible promoters. These plasmids were characterized over a range of inducer concentrations, in the presence of non-cognate inducer molecules, and with several growth media, and their characteristics were documented in a standard format datasheet. A three plasmid system was used to investigate the impact of multiple origins of replication on plasmid copy number.

Conclusions: The standardized collection of vectors presented here allows the user to rapidly construct and test the expression of genes with various combinations of promoter strength, inducible expression system, copy number, and antibiotic resistance. The quantitative datasheets created for these vectors will increase the predictability of gene expression, especially when multiple plasmids and inducers are utilized.

Figure 1

Schematic diagram of the BglBrick part assembly. Four unique restriction sites (EcoRI, BglII, BamHI, and XhoI) are used for the BglBrick standard assembly. KAN is the kanamycin resistance marker.

Figure 2

Plasmid design and nomenclature of BglBrick plasmids (pBb). (A) Plasmid design of pBb vectors. The plasmid is composed of three modules: antibiotic resistance gene module, replication origin module, and expression module, which includes the repressor, promoter, gene of interest (rfp or gfp), and terminator. BglBrick sites are in red boxes. (B) Nomenclature of the pBb vector system. The identity of the vector is described by three letters containing the information of replication origin, promoter, and antibiotic resistance marker as indicated. The prefix pBb is used for BglBrick plasmids and the protein gene name in the plasmid is included at the end of the vector description.

Figure 3

Datasheet for the pBbE5 vector. The datasheet includes a general description of BglBrick vector and a summary of its expression properties.

Figure 4

BglBrick plasmid copy numbers in DH1 and BLR(DE3). The blue bars are for DH1 strains, and the purple bars are for BLR(DE3) strains. The dark colored bars are for the single-plasmid strain, and the light colored bars are for the three-plasmid strain containing pBbA8a-CFP, pBbE5c-YFP and pBbS2k-RFP. Plasmids with the pBBR1 origin were not tested in the three-plasmid strain. (A) Plasmid copy number for single plasmid strain (B) DH1 plasmid copy number comparison for single and three plasmid strain (C) BLR(DE3) plasmid copy number comparison for single and three plasmid strain.

Figure 5

Expression of three different proteins from a single strain at various inducer concentrations. pBbA8a-cfp, pBbE5c-yfp and pBbS2k-rfp were transformed in E. coli BLR(DE3) and the fluorescent proteins were expressed under various inducer concentration combinations.