Details

Autor(en) / Beteiligte

• Endy, Drew
• Chan, Leon Y
• Kosuri, Sriram

Titel

Refactoring bacteriophage T7

Ist Teil von

• Molecular systems biology, 2005, Vol.1 (1), p.2005.0018-n/a

Ort / Verlag

Chichester, UK: John Wiley & Sons, Ltd

Erscheinungsjahr

2005

Link zum Volltext

Quelle

Electronic Journals Library

Beschreibungen/Notizen

• 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 redesign 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 initial design goals were to physically separate and enable unique manipulation of primary genetic elements. Implicit in our design are the hypotheses that overlapping genetic elements are, in aggregate, nonessential for T7 viability and that our models for the functions encoded by elements are sufficient. To test our initial design, we replaced the left 11 515 base pairs (bp) of the 39 937 bp wild‐type genome with 12 179 bp of engineered DNA. The resulting chimeric genome encodes a viable bacteriophage that appears to maintain key features of the original while being simpler to model and easier to manipulate. The viability of our initial design suggests that the genomes encoding natural biological systems can be systematically redesigned and built anew in service of scientific understanding or human intention. 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. 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. 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. We redesigned the genome of bacteriophage T7 in order to specify an engineered surrogate that is easier to study, understand, and extend. We replaced the left 11 515 bp of the wild‐type genome with 12 179 bp of engineered DNA. The resulting chimeric phage are viable. Synthetic genomes that encode only our current understanding of natural biological systems should facilitate discovery science—for example, differences between the encoded behavior of a synthetic and natural genome can serve to highlight relevant gaps in our knowledge. Or, synthetic genomes can be used to construct engineered surrogates whose designs are optimized for human purposes—for example, ease of understanding and manipulation.