T7.2

T7.2 is part of a larger project of Rebuilding T7 to construct more understandable model organisms. T7.2 is the second iteration of constructing a physical instance our understanding of T7, which began with T7.1.

Background

The T7.1 genome was more constrained by our initial uncertainty than driven by our primary design goal – to construct a genetic system that is a physical instance of our understanding of a biological system that we believe, to first approximation, to understand. While we will complete construction of T7.1, and will make use of T7.1 as an intermediate tool for comparison of wild type to T7.2, T7.1 is not best suited for our proposed work. Stated differently, we don’t care about the exact sequence of the wild-type T7 genome per se. Instead we care about the system encoded by the wild-type T7 genome; the wild-type genome may be one instance of many possible genomes encoding equivalent systems. Here, we are working to test the hypothesis that our current understanding of the genetic elements encoded on the wild-type genome is sufficient to specify a new genome that encodes a recapitulation of the wild-type system’s performance. There’s a small chance that, many years from now, as we continue to better understand the intricacies of T7 development and nature’s designs, we will end up with T7.N, where N is a large positive integer, and find that T7.N has the same sequence as the original isolate, at which point we’ll understand why.

Goals

We are interested in how the parts of biological system are organized onto a genome to produce system-level behavior. The primary purpose of T7.2 is to construct a biological organism that is easier to model than the original biological isolate. There are existing aspects of the natural isolate that make it particularly well suited to system-level analysis. We want to make every effort to preserve these aspects, which include:

1. Viability --
2. Existing knowledge of the functional genetic elements --
3. Relatively decoupled from host physiology --
4. Coupling of entry and transcription, leading to a natural organization on T7 genome -- This makes it easier to model to some extent.

However, we feel the original biological isolate is not necessarily where we should begin to understand how system-behavior is produced. Specifically, in designing T7.2, the following goals will drive our design; the first three goals revisit or extend those used in the design of T7.1.

1. Our design of T7.2 will enable the unique and selection-independent manipulation of each genetic element via restriction enzymes.
2. We will specify a genome that does not include any functions that might be encoded via the physical coupling of multiple genetic elements.
3. We will specify a genome that only includes elements that we believe contribute to phage gene expression. Moving beyond our design of T7.1, we will actively erase or delete elements of unknown function. In addition, efforts will be made to made to remove unknown genetic elements.
4. To attempt to make our modeling of gene expression easier, we will use standard synthetic elements in place of the natural elements that regulate transcription and translation.
5. We will make efforts to add reporters and make the genome more amenable to measurements.

Taken together, our design of T7.2 should specify a genome that is simpler to model and manipulate, in which we have a putative function for each base pair of DNA involved in phage gene expression. Thus, we hypothesize that T7.2 will also encode a dynamic system that is easier to model and interact with, relative to the wild type.