T7.1

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Background

Wild-type T7 is a superb organism for discovering the primary components of a natural biological system. However, our experience indicates that the original T7 isolate is not best suited for understanding how all the parts of the phage are arganized to encode a functioning whole. We decided to engineer a surrogate genome, which we designated T7.1, that would be easier to study and extend.

Goals

1. We wanted to insulate and enable independent manipulation of all identified genetic elements.
2. We wanted the T7.1 genome to encode a viable bacteriophage; at the start of this work, we were uncertain how many simultaneous changes the wild-type genome could tolerate.

Method

1. Reannotation of the wild-type T7 genome, thus defining the functional genetic elements
2. Specification of T7.1 genome design and sequence
3. Construct sections individually
4. Construct chimeric phages that contain replace a single wild-type section with a rebuit section
5. Combine sections of rebuilt phage into a single rebuilt phage
6. Characterize chimeric phage

Progress

1. Reannotation -- The wild-type T7 genome is a 39,937 base pair linear double-stranded DNA molecule. We annotated the genome by specifying the boundaries of the following functional genetic elements: 57 open reading frames, 57 putative RBSs encoding 60 proteins, and 51 regulatory elements controlling phage gene expression, DNA replication, and genome packaging. A genbank file of the reannotation can be found here.
2. Specification of T7.1 genome -- The designed sequence of T7.1 can be found here.
   • Briefly, to specify the architecture of T7.1, we organized the functional genetic elements into 73 ‘parts.’ Each part contains one or more elements. While the DNA sequence of elements within parts may overlap, there is no overlap across part boundaries. Next, we organized contiguous parts into ‘sections’ with section boundaries defined by restriction endonuclease sites found only once in the sequence of the wild-type genome. Six sections, alpha through zêta, make up the T7.1 genome. Sections were used to compartmentalize changes across the genome. In addition, sections can be built, tested, and manipulated independently.
   • To specify the DNA sequence of T7.1, we eliminated sequence overlap across part boundaries. Overlaps were eliminated by exact duplication of the wild-type DNA sequence; subsequent sequence editing produced a single instance of any duplicated element. All sequence edits within open reading frames were silent and maintained the wild-type tRNA specification or, when necessary, specified a higher abundance tRNA (Ikemura, 1981). We also added bracketing restriction endonuclease sites to insulate and enable the independent manipulation of each part. Bracketing sites are not used elsewhere in the sequence of any one section but are reused across sections. The DNA sequence of T7.1 changes or adds 1,424 base pairs to the wild-type genome.