Rebuilding T7

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Summary

Wild-type T7 is a superb organism for discovering the primary components of a biological system [Studier, 1972]. However, is the original T7 isolate also best suited for understanding how all parts of the phage are organized to encode a functioning whole?

Motivations

Physical Model to further understand biology

We are interested in questions of how the genetic components of an organism are organized on the genome to carryout functions such as development even in the face of significant environmental variations. However, to rigorously answer these questions, we need to first understand how changes in genome organization lead to changes in the timing and level of gene expression. However our efforts to create empirical computational models based on observations of the primary genetic elements and mechanisms governing gene expression have fallen short of providing system-level predictive capacity. It is unclear whether the differences between our measurements and models are due to our inability to parameterize and simulate the complex biophysical processes involved, or our understanding of the biophysical processes is just incorrect or incomplete. Moving forward, instead of further studying wild-type T7, we can try encoding our understanding of the biophysical processes onto a new genome. We will try to only encode those functions which we understand, while actively removing those that we don't. Differences between the behavior of the engineered and wild-type phage will highlight gaps in our understanding, and will lead to follow-on science.

New Model Organism

On the other hand, our interest in T7 is somewhat arbitrary. If we can construct a better model organism that will let us study hypotheses of how the organization of genetic components make up a biophysical system, it may not be necessary to relate that back to the wild type.

T7 Versions

T7.1

At the start of this work, we were uncertain how many simultaneous changes the wild-type genome could tolerate. Hence we were conservative with our original design. Two goals drove our design of T7.1. First, we wanted to insulate and enable independent manipulation of all identified genetic elements. Second, we wanted the T7.1 genome to encode a viable bacteriophage.

T7.2

Five goals will drive our design of T7.2; the first four goals revisit or extend those used in the design of T7.1. First, we will specify a genome that only includes elements that webelieve contribute to phage gene expression. Moving beyond our design of T7.1, we will actively erase or delete elements of unknown function. Second, we will specify a genome that does not include any functions that might be encoded via the physical coupling of multiple genetic elements. Third, our design of T7.2 will enable the unique and selection-independent manipulation of each genetic element via restriction enzymes. Fourth, for practical reasons, our design of T7.2 must encode a viable bacteriophage. Fifth, 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. Taken together, our design of T7.2 should specify a genome that is simpler to model andmanipulate, 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 dynamicsystem that is easier to model and interact with, relative to the wild type.

People

Leon Chan, Sriram Kosuri, Drew Endy