Refactoring bacteriophage T7

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Leon Y Chan^1,a, Sriram Kosuri^2,a & Drew Endy²

^aThese authors contributed equally to this work

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Synopsis

Natural biological systems are selected by evolution to continue to exist and evolve. Evolution likely gives rise to complicated systems that are difficult to understand and manipulate. Here, we redesigned the genome of a natural biological system, bacteriophage T7, in order to specify an engineered surrogate that, if viable, would be easier to study and extend.

Our work was initially motivated by past failures in modeling T7 development and by a desire to better understand how the parts that comprise bacteriophage T7 work together to encode a functioning whole (Kirschner, 2005). The approach we used was inspired by the practice of 'refactoring,' a process that is typically used to improve the design of legacy computer software (Fowler et al, 1999). In general terms, the goal of refactoring is to improve the internal structure of an existing system for future use, while simultaneously maintaining external system function.

Six specific goals drove our redesign of a new T7 genome, which we designated T7.1. First, we wanted to define a set of components that function during T7 development and, for each element, choose an exact DNA sequence that we could use to encode element function. Second, we wanted the DNA sequence encoding the function of any one element to not overlap with the DNA sequence encoding any other element. Third, we wanted the DNA sequence of each element to encode only the function assigned to that element and not any other functions. Fourth, we wanted to enable the precise and independent manipulation of each element. Fifth, we needed to be able to construct the T7.1 genome. Sixth, we needed the T7.1 genome to encode viable bacteriophage; at the start of this work, we were uncertain how many simultaneous changes the wild-type genome could tolerate. Figure 1 details the sorts of DNA sequence changes we made during the refactoring process.

Figure 1

Element decompression and part design. (A) The coding regions of genes 2.8 and 3 overlap in the wild-type T7 genome. The RBS of gene 3 (underlined) is encoded within gene 2.8. (B) Distinct genetic parts make up the T7.1 genome. The natural RBS and start codon (green) for gene 3 are disrupted by point mutations (capitals); mutations do not change the amino-acid sequence of the 2.8 protein. Parts 28 and 29 are separated by bracketing restriction sites, BamHI (blue) and EagI (orange). Supplementary Figure S3 lists all changes in the DNA sequence of T7.1 relative to wild-type T7.

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We split the design of the T7.1 genome into six sections that can be built and tested independently (Figure 2). We constructed the first two sections, alpha and beta. Alpha and beta replace the left 11 515 bp of the wild-type genome with 12 179 bp of engineered DNA, and encode the entire T7 early region, the primary origins of DNA replication, most of the T7 middle genes, and the control architecture that regulates T7 gene expression. We combined alpha and beta with the remainder of the wild-type (WT) genome to produce three chimeric phages: alpha-WT, WT-beta-WT, and alpha-beta-WT. We tested and recovered viable chimeric phage by transfection and plating. All three chimeric phages are viable (Figure 4). We isolated DNA and performed restriction digests across alpha and beta to confirm that individual parts could be independently manipulated.

Figure 2

Genome design. (A) We split the wild-type T7 genome into six sections, alpha through zêta, using five restriction sites unique across the natural sequence. (B) Wild-type section alpha genetic elements: protein coding regions (blue), RBSs (purple), promoters (green), RNaseIII recognition sites (pink), a transcription terminator (yellow), and others (gray). Elements are labeled by convention (Dunn and Studier, 1983). Images are not to scale, but overlapping boundaries indicate elements with shared sequence. The five useful natural restriction sites across section alpha are shown (black lines). (C) T7.1 section alpha parts. Parts are given integer numbers, 1–73, starting at the left end of the genome. Unique restriction site pairs bracket each part (red/blue lines, labeled D[part #]L/R). Added unique restriction sites (purple lines, U[part #]) and part length (# base pairs, open boxes) are shown. We do not know if sequence changes in and around parts 6 and 7 destroy the minor E. coli promoter, B. (D) Wild-type section beta genetic elements. (E) T7.1 section beta parts. Supplementary Figure S2 depicts the six sections, alpha through zêta, which make up the T7.1 genome.

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Figure 4

Characterization of T7.1 (A) Lysis of log-phase liquid cultures of E. coli BL21 (30°C) by wild-type T7 (black), alpha-WT chimera (red), WT-beta-WT chimera (blue), alpha-beta-WT chimera (orange); absorbance of 0.275 is 2E8 cells/ml. Vertical bars show standard deviation at each time point (based on four replicates) (Supplementary information). (B) T7 plaques on E. coli BL21 (24 h, 37°C, 10 cm Petri dish). Clockwise from top left: wild-type (WT) T7, alpha-WT chimera, WT-beta-WT chimera, alpha-beta-WT chimera (Supplementary information).

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We constructed sections alpha and beta manually. Recent advances in de novo DNA synthesis technology have enabled the rapid automatic synthesis of DNA fragments the size of the T7.1 genome sections (Stemmer et al, 1995; Yount et al, 2000; Kodumal et al, 2003; Smith et al, 2003; Tian et al, 2004). Continued improvements in DNA synthesis technologies will directly accelerate the engineering of biology, and impact the science of biology at least as much as large-scale automated DNA sequencing technology (Carlson, 2003).

Our work with T7 suggests that the genomes encoding other natural, evolved biological systems could be redesigned and built anew in support of scientific discovery or human intention. For systems beyond model laboratory organisms, pursuing such work will require the widespread societal acceptance of responsibility for the direct manipulation of genetic information.

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