T7.2

T7.2 is part of a larger project of Rebuilding T7 to construct a more understandable model organism. T7.2 is the second iteration of our work in creating a more modelable organism, which began with T7.1.

Background

The T7.1 genome was more constrained by our initial uncertainty on the number of simultaneous changes T7 could tolerate than driven by our primary design goal – to construct a genetic system to help us understand how parts of a genetic system act in coordination to produce system-level behavior. 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.

Goals

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 -- Here viability refers to the ability to propagate the new species for practical experimental purposes.
2. Existing knowledge of the functional genetic elements -- We must continue to harness the tremendous amount of knowledge gained from genetic and biochemical experiments of T7 over the past 60 years. To some extent, these studies are what allow our current models to as detailed as they are.
3. Relatively decoupled from host physiology -- T7 begins shutting off host transcription and solublizing the host genome within minutes of infection. There are very few host proteins necessary for T7 infection. We do not want to purposely or incidentally want to increase the dependency of the phage upon the host so that we can minimize modeling the intricacies of host physiology.
4. Coupling of entry and transcription -- This coupling leads to the natural organization of genes on the T7 genome. In addition, the timing of genome entry, to some extent, makes modeling the phage easier. In addition, any importance of the ordering of elements on the natural isolate will be more relevant to T7.2.

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 five goals will drive our design; the first three goals revisit or extend goals motivating 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.
   1. reduced gene sets?
   2. codon shuffling?
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 a genome that is more anemenable to measurements that are important to us, such as adding reporters of transcription and translation.

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. We hypothesize that as a result of the more parsimonious genome design, T7.2 will also encode a dynamic system that is easier to model and interact with, relative to the natural biological isolate.

Meta Considerations

Standardization

We want to standardize certain functional genetic elements to make them easier to model. For example, instead of further characterizing every different ribosome binding site and promoter, we can standardize on a set of that would take considerably less effort to characterize.

Measurement

We want to increase the ability to measure different aspects of phage biology. This may including mRNA and protein reporters, and/or optimizing DpnI restriction sites to ease entry assays.

Manipulability

We need to increase our ability to make selection-independent changes to the genome easily. We will take the some of the lessons we learned in T7.1 about the types and distribution of restriction sites needed.

Encoding

We want to reduce, to as large of an extent as possible, the number of genetic elements that are included in T7.2 that do not encode functions that we do not know about.

Functional Genetic Element Design Considerations

Coding Domains

We would like to remove a number of genes which we have reason to believe encode no observable function. The following genes are unconserved across other T7 likes and encode no known function during T7 developement: 0.4, 0.5, 0.6A/B, 1.4, 1.5, 1.6, 1.8, 2.8, 3.8, 4.1, 4.2, 4.7, 5.3, 5.9, 6.3, 7, 7.7. We will begin by removing these genes first.

There is a larger subset of genes that are thought not to encode functions during T7 development, but are conserved across other T7 genomes. We will continue to keep these in the genome, but it make easier to delete them in the future. These include genes 0.3, 1.1, 4.3, 4.5, 5.5-5.7, 5.7, 6.5, 18.5, 18.7, 19.2, 19.3, 19.5.

Ribosome Binding Sites

We will use a standardized set of ribosomes known to have different levels of expression. We will replace the natural sites with sites that are encoded by the BioBrick part numbers:
BBa_B0034 -- strongest 1
BBa_B0030 -- strong 0.6
BBa_B0032 -- medium 0.3
BBa_B0031 -- weak 0.07
BBa_B0033 -- weaker 0.01

We will assign one of these parts to each coding domain based on both knowledge of the amounts of total protein production necessary, as well as computational analysis of the existing ribosome binding sites strength.

Host Promoters

We will encode only the the 3 strong host promoters, A1, A2, and A3. We will actively take steps to remove the other host promoters identified in the annotation. Finally, we will keep the boxA anti-termination site intact. It is worth considering if we should remove all but one of the T7 promoters, such as A1.

Phage Promoters

We want to standardize the phage promoters that we encode onto the genome. In our models, we split the promoters into three class; weak binding and processivity, weak processivity, or strong promoters. The natural T7 promoters on the T7 genome are listed below, along the classification they fall into. The promoters in bold are the canonical promoter in each class that have the most kinetic studies associated with them. Thus we will use the three promoters in bold as the prototype sequences in each class that we will standardize the other promoters to.
øOL -- weak processivity (-11,1)
ø1.1A -- weak binding & processivity (-17,2,4)
ø1.1B -- weak processivity (3,4,5)
ø1.3 -- weak binding & processivity (-5,5)
ø1.5 -- weak processivity (-2,3,4,5)
ø1.6 -- weak processivity (-2,3,4,5,6)
ø2.5 -- weak processivity (-1,1,4,5)
ø3.8 -- weak binding & processivity (-13,-12,-11,-2)
ø4c -- weak binding & processivity (-17,-13,2)
ø4.3 -- weak processivity (-2,3,4,5,6)
ø4.7 -- weak binding & processivity (-17,-16,-13,3,4,5,6)
ø6.5 -- strong
ø9 -- strong
ø10 -- strong
ø13 -- strong
ø17 -- strong
øOR -- strong

Terminators

We will keep our defiinitions for the terminators as they were in T7.1. Namely, TE and Tø will remain intact, while effort will be made to not alter the CJ terminator.