T7.1
From OpenWetWare
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
- We wanted to insulate and enable independent manipulation of all identified genetic elements.
- 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
- Reannotation of the wild-type T7 genome, thus defining the functional genetic elements
- Specification of T7.1 genome
- Specification of parts, which can contain one or more functional genetic elements, along with defining precise boundaries for these parts. While the DNA sequence of elements within parts may overlap, there is no overlap across part bondaries.
- We organized these parts into sections, whose boundaries are defined by restriction endonuclease sites found only once in the sequence of the wild-type genome. Sections were used to compartmentalize changes across the genome, and so that each section can be built and tested independently.
- Construct sections individually
- Construct chimeric phages that contain replace a single wild-type section with a rebuit section
- Combine sections of rebuilt phage into a single rebuilt phage
- Characterize chimeric phage
Progress
- 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
