Bacteriophage-based synthetic biology for the study of infectious diseases

Abstract

Since their discovery, bacteriophages have contributed enormously to our understanding of molecular biology as model systems. Furthermore, bacteriophages have provided many tools that have advanced the fields of genetic engineering and synthetic biology. Here, we discuss bacteriophage-based technologies and their application to the study of infectious diseases. New strategies for engineering genomes have the potential to accelerate the design of novel phages as therapies, diagnostics, and tools. Though almost a century has elapsed since their discovery, bacteriophages continue to have a major impact on modern biological sciences, especially with the growth of multidrug-resistant bacteria and interest in the microbiome.

Figure 1. Bacteriophage-derived parts can perform molecular computation in microbial cells

(A) Truth table for AND gate function. An AND gate has a TRUE output only when both inputs are TRUE. (B) Split T7 RNA polymerase can act as a transcriptional AND gate. Inputs A and B, which can be exogenous signals such as small-molecule inducers or endogenous signals, control expression of the N-terminal and C-terminal halves of T7 RNA polymerase, respectively, such that simultaneous expression leads to a reconstituted polymerase that can drive production of the output gene [56]. (C) Bacteriophage-derived recombinases Bxb1 and phiC31 implementing AND gate functionality with integrated memory. Recombinase activity leads to inversion of the intervening terminator sequence flanked by recognition sites. Activation of Bxb1 and phiC31 leads to inversion of two unidirectional terminators, such that RNA polymerase can drive expression of the output gene [22].

Figure 2. Phage-Assisted Continuous Evolution

In phage-assisted continuous evolution (PACE), the protein activity of interest is linked to expression of the M13 coat protein pIII, which is required for binding and initiation of the phage infection cycle [73]. Within infected cells (top), the protein of interest is expressed from the injected M13 genome and successful target activity drives expression of gene III on the accessory plasmid, permitting the assembly of infectious phage progeny that can be further amplified and selected through subsequent rounds of infection (left). In the example shown here, the protein of interest is T7 RNA polymerase (T7 RNAP), which can successfully drive production of gene III only if it recognizes the promoter that controls gene III expression, thus enabling the evolution of T7 RNA polymerase variants that can target new promoter sequences. If activity is insufficient to drive expression of the coat protein, progeny will be non-infectious and fail to amplify. The target protein is evolved in a continuous fashion in the “lagoon” (bottom), where the encoding infectious phages continually amplify via the input of fresh cells, while non-infectious particles fail to infect new cells and are removed in the outflow.

Figure 3. Bacteriophage can modulate target cells by delivering genetic payloads

Bacteriophage such as the temperate phage Lambda (A) and the filamentous phage M13 (B) have been used to deliver non-native DNA to target cells. (A) Delivery of dominant sensitive genes encoding wild-type enzymes such as gyrA and rpsL results in the resensitization of a cell to antibiotics to which resistance had previously been conferred by mutations in these genes [89]. (B) Delivery of transcription factors can modulate regulatory networks in bacteria, such as the SOS response normally induced in order to respond to cellular stress and repair damaged DNA [90]. The dominant LexA3 variant can inhibit the SOS response and resensitize cells to some antibiotics as well as reduce the number of emerging resistant cells.

Figure 4. Synergy between synthetic biology and bacteriophage

New tools and techniques from synthetic biology have enabled phage research and the development of novel therapies. Likewise, bacteriophage components have fueled innovation in synthetic biology.