T7.2/Design

T7.2 Alpha Design

Design

An initial draft design of T7.2

Based on the gene, RBS, RNaseIII, and promoter classifications described below, I have constructed a draft design shown in the thumbnail to the right. This is the component level specificaiton. Further refinement will be needed at the sequence level (DpnI optimization, restriction sites, codon shuffling, and how much extra DNA we will have to add).

Comments

Sri Kosuri 19:17, 28 March 2006 (EST): I'm wondering what to do when there are there are elements removed that cause two promoter elements to be right next to each other. For example, in this initial design, ø1.5 and ø1.6 (standardized to ø2.5) are right next to each other because we are planning to eliminate 1.5.

Sri Kosuri 19:35, 28 March 2006 (EST): By the definition of what coding domains we are keeping and removing (below), I think we should probably get rid of 0.3. Is there any other assignable function for this gene other than host restriction evasion?

Functional Genetic Element Design Considerations

Coding Domains

A number of the CDS on the natural T7 genome that do not contribute to phage development in the set of conditions that we wish to assay them, and thus confound our systems-level analysis. 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 development: 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 we are interested in removing, but will continue to keep these in the genome for a variety of reasons (evolutionary conservation, contribution to development unclear). We will it make easier to delete them in the future. This subset 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.

For details on individual genes, check out the gene specification page.

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.

For details on individual genes, check out the gene specification page.

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.

Removed Host Promoters

B: Located mostly in gene 0.5, which is not encoded
C: Only -35 region is included within coding domain of gene 0.7; no further mutation necessary
E: Strong -10 region mutated, -35 region not included
S1: -35 and -10 regions edited to remove promoter
S2: -35 region edited, -10 region already nonoptimal
S3: -35 and -10 regions edited to remove promoter
S4: -35 and -10 regions edited to remove promoter
S5: Located within gene 5.3 which is not included
S6: -35 and -10 regions edited to remove promoter
F1: Only -35 region is included within coding domain of gene 6.5; no further mutation necessary
F2: -35 region mutated, -10 region already nonoptimal

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. TE and Tø will remain intact, while effort will be made to not alter the CJ terminator.

Other Changes

DpnI optimization

• Word file proposal for DpnI remapping
• PDF map of resulting restriction pattern
• Excel file containing sites on DNA to be changed
• Excel file containing graphical depiction of restriction fragments