Bacteriophage T7.2 -- design optimization
Design of T7.2 with Venus YFP and removed secondary structure for strong RBS overlap.
Gene 0.7 codes for a protein that inactivates host-catalyzed transcription, it is also a serine-threonine protein kinase. The two activities are separable. Gp0.7 phosphorylates many host proteins, including ribosome components, ribonucleases III and E, and the β and β?? subunits of E. coli RNAP. The BR3 and Y49 strains contain the rpoC-E1158K mutation, which renders gp0.7 essential for phage growth. The mutation affects the region of β?? to which the T7 protein gp2 binds. Gene 0.7 is non-essential in wild-type hosts and is actually detrimental when T7 is grown at 30˚C in rich media. Early T7 RNA and protein synthesis is prolonged after infection by gp0.7 mutants. However, phage growth at elevated temperatures or in minimal media, or in cells harboring the Col Ib plasmid renders gene 0.7 essential. The processes underlying the conditional necessity for gp0.7 function have not been determined. The E. coli C promoter -35 region is located on the 3' end of the coding sequence. Removing the downstream region will probably be sufficient to delete functioning of the promoter.
Encodes gp1: T7 RNA polymerase from wild-type T7; Suppressor mutation in 19 allows no gene 1 late in infection. It's thought that gene 1 is hanging out at the CJ terminator at the pause site... recruiting 18 and 19 after packaging. (Studier FW, PC)
open reading frame for gene 1.1. from wild-type T7 genome
Gene 1.2 inhibits E. coli dGTPase and converts it into a rGTP-binding protein, which is important for T7 only when dgt is over-expressed (optA1 mutation). T7 gene 1.2 also causes phage exclusion by the F plasmid, but T3 gene 1.2 overcomes the exclusion system.
Encodes for a endonuclease that prefers ssDNA. It functions along with gene 6 to degrade the host chromosome during infection and provide nucleotides for subsequent phage DNA replication. Gene 3 is also a Holliday junction resolvase.
Gene 3.5 codes for lysozyme, which is actually not a lysozyme but an N-acetylmuramyl-L-alanine amidase. The most important functions of lysozyme are also not in cell lysis but lie in controlling initiation and termination by T7 RNAP, which in turn affect DNA replication and packaging.
The full-length gene 4 protein is a 5??->3?? helicase of the E. coli DnaB family and a DNA primase. An in-frame internal start site within gene 4 results in a protein, gp4B, that lacks the N-terminal 63 amino acids of the 63 kDa gp4A, and consequently lacks primase activity. The primase recognition sites on ssDNA are 5??-G/TGGTC, and 5??-GTGTC.
mutation from T7 to remove RBS from gene 2.8
Sequence comes from the natural gene 5 RBS. Using gene 5 as a strong RBS is from initial results from experiments characterizing all natural T7 RBSs.
Sequence comes from the natural B0030. Using B0030 as a medium strength RBS is from initial results from experiments characterizing all natural T7 RBSs.
Contains 9 bp before and 10 bp after stem loop(up to start of 0.3 RBS).
The ATP-dependent DNA ligase is only essential in E. coli lig mutants, but its presence enhances phage growth in wild-type hosts.
Gp2 binds to and is an inhibitor of E. coli RNAP but in K-12 or B strains the defects due to a lack of gene 2 activity are reduced DNA replication and premature breakdown of replicating DNA, specifically at the left genome end where the major E. coli promoters are found.
structural tail protein of phage capsid; 12 copies per virion
a structural tail protein of the phage capsid; 6 copies per virion
internal core protein of phage capsid; ~8 copies per virion
Internal core protein of phage capsid; ~4 copies per virion
large terminase subunit. binds capsid pro-head. Also contains within the sequence for 19.2 and 19.3 within the gene.
lysis associated protein. Homologs of 18.5/18.7 are found in all phages (in T1 the two proteins are fused); contains gene 18.7 within coding domain.
Bases have been changed to remove gene 1.8 start site acataaAgataaatgttaCgcataa
Mutations from wild-type on 3' end to mutate Gene 14 RBS
Mutations on 3' end to remove Gene 15 RBS
3' end of gene mutated to remove gene 0.4 RBS
Takes 6bp ahead of the expected start of the stem loop, and 3bp after the expected termination site (11bp after end of stem loop). Causes termination by E. coli RNA polymerase (but not T7 RNA polymerase).
Left end of T7 genome. Includes terminal repeat, short repeats, leftward host promoter A0. Contains sequence up to -27bp of T7 promoter phi0L.
Right End of T7 DNA. Important in packaging. Contains short terminal repeat region and the right terminal repeat. Contains sequence starting after gene 19.5 stop site.
Host promoter region containing 3 promoters and boxA antitermination site. Takes natural sequence from end of phi0L to start of R0.3
Venus Sequence obtained from Prof. Miyawaki. Codon optimized from Gene Design by Boeke Lab.
Head protein. ~18 copies per virion. Gp6.7 is not absolutely essential, morphological normal T7 particles are produced in its absence but have reduced infectivity.
Gene 7 was originally defined by mutations that mapped between genes 6 and 8 but was subsequently shown to be two genes, 7 and 7.3. Gp7.3 is a tail protein, present in ~30 copies per particle, that is essential for infectivity though not particle formation.
Head-tail connector. 12 copies per virion. Three possible host promoters with -35 regions at 293, 1100, 1249
major/minor capsid protein. The 10B portion is non-essential, 10A-only phages seem to grow normally. ~430 copies of 10A and 16 copies of 10B in capsid (415 expected from symmetry). JJ Dunn removed various pieces of terminator.. if you remove hairpin, the gene 10 RNA is obliterated. Also, got rid of gene 10 pre-shine dalgarno and could not get translation off gene 10. (Dunn JJ, PC)
T7 tail fiber protein of phage capsid (adsorption); 14 +/- 3 (eighteen expected by symmettry) copies per virion; 3 copies per tail fiber
3' end mutated to remove gene 6.3 startsite and alter ribosome binding site
Mutations on 3' end to remove gene 18 RBS
silent mutation at 3' end to remove gene 6.7 RBS. -35 region of F1 probably non-functional at 5' end of gene.
Removes the overlap of gene 5.9 RBS. Not the actual sequence in the design of T7.1, allows the frameshift again.
Taken 10 bp before expected start of stem loop structure, and 3bp after expected stop of transcription (7bp after stem loop)
T7 DNA Polymerase consists of two proteins, the gene 5 protein and the processivity factor E. coli thioredoxin. Thioredoxin forms a 1:1 complex with the T7 protein and increases its processivity by at least 1000-fold, the complex also increases the activity of the associated 5??->3?? exonuclease on dsDNA but has little effect on ssDNA exonucleolytic activity.
Conversion of the iGEM parts registry to SBOL2.1