Our contributions are to sort out the genome information of T4 phage, replenish the gene information of phage T4 genome according to the latest results (More details are in the appendix) and made a new genome annotation map, which provides accessible and reliable data for later researchers to reboot engineered phages.
1 Our First Contribution
Figure 1. Interactive genome annotation.
An interactive figure is shown above in which you can easily get access to the information of T4 phage genes in hover position. If the figure above doesn't work, click the link to view the interactive version: https://2020.igem.org/Team:DUT_China/T4_phage
The information are essential gene names, position, length, weight and product in this figure. We use colors of blue, purple, orange and yellow to represent the part responsible for encoding tail structure, head structure, energy control system and central dogma system, respectively.
Through genome annotation above, our phage reboot technology can be applied with convenience. By this, future researchers can easily segment the phage genome according to whether it is conservative or not, and add the variable and non-variable parts into different plasmids. Only the plasmids with variable parts (such as knockout gene) are edited, and then researchers can copy these plasmids through yeast cells and then transfer them into E. coli. And in the next step, phages reboot. Therefore, original complex phage genome editing (through gene recombination or non-cellular system) can be innovatively transformed into simple plasmid editing.
3 Our Third Contribution
Our third contribution is to replenish relevant information according to the latest results (More details are in the appendix) and made sequences alignment (Fig.2) to compare 4 kinds of E. coli phages, T4, T6, slur03, KIT03.
Figure 2. Bioinformatic analysis of phages DNA sequences alignment.
Our sequences alignment result has revealed relationships between T4, T6, slur03, KIT03. Five kinds arrows with different colors reveal different essential functions, which gives us an evidence to ensure our judgement.
And in sequences alignment above:
1.Only sequence similarities more than 100 base pairs are listed here.
2.The possible functions of T6, KIT03 and slur03 phages sequence fragments are inferred from T4.
3.Expectation value is 0.001.
4.The scoring matrix used here is BLOSUM62.
5.Here we use Easyfig software to draw the graph above[1].
5 Appendix
1. SegD
A novel phage T4 endonuclease, SegD, recognizes an extended, 16 bp long, site, cleaves it asymmetrically to form 3′-protruding ends and digests both unmodified DNA and modified T-even phage DNA with similar efficiencies.
Homing endonucleases are a group of site-specific endonucleases that initiate homing, a nonreciprocal transfer of its own gene into a new allele lacking this gene. Endonuclease SegD is homologous to the GIY-YIG family of homing endonucleases. However, SegD was not able to initiate homing of its own gene[6].
2. RNase E
Early gene expression in bacteriophage T4 is controlled primarily by the unique early promoters, while T4-encoded RegB endoribonuclease promotes degradation of many early messages contributing to the rapid shift of gene expression from the early to middle stages.
The initial cleavage by RegB of phages TuIa and RB69 enables degradation of early phage mRNAs by the major Escherichia coli endoribonuclease, RNase E[7].
3. g23(capsid gene)
Genomic information is preserved among viral subsets and can be used for phylogenetic classification of viruses and for evaluation of viral diversity in the environment. The capsid gene of T4-type bacteriophages, g23, is the most widely applied gene for evaluating the diversity of the T4-type bacteriophage family[8-11].
4. gp4
gp4 (phage T4 gene product 4) plays an essential role in the head-tail joining step of T4-like phages. Amber mutations in gene 4 prevent heads and tails from joining efficiently and 50% of the heads produced lack DNA[12].
5. UvsY(recombination mediator protein)
Bacteriophage T4 UvsY is a recombination mediator protein that promotes assembly of the UvsX-ssDNA presynaptic filament. UvsY helps UvsX to displace T4 gene 32 protein (gp32) from ssDNA, a reaction necessary for proper formation of the presynaptic filament.
UvsY interacts strongly with stretched DNA in the absence of other proteins. In the presence of gp32 and *I, UvsY is capable of strongly destabilizing gp32-DNA complexes in order to facilitate ssDNA wrapping, which in turn prepares the ssDNA for presynaptic filament assembly in the presence of UvsX. Thus, UvsY mediates UvsX binding to ssDNA by converting rigid gp32-DNA filaments into a structure that can be strongly bound by UvsX[13].
6. The baseplate protein gene product
The proteins with known atomic structures: gp 5, gp8, gp9, gp11, gp12, and gp27.
The proteins whose structures had not yet been established: gp6, gp7, gp10, gp25, gp29, gp48, gp53, and gp54.
During the attachment of the phage to the host, the baseplate changes its conformation from being a hexagonally shaped dome into a planar six-pointed star structure. This conformational change triggers contraction of the sheath, which results in penetration of the cell envelope by the tail tube, allowing the phage to transfer its DNA into the host cell[14].
7. holin protein (T); antiholin (RI)
In T4 phages, the length and fecundity of the infection cycle are determined by small membrane proteins called holins and their specific regulators, the antiholins.
Holins initiate lysis by permeabilizing the cytoplasmic membrane at a genetically programmed time, setting in motion a lysis pathway that results in release of the viral progeny.
Free periplasmic RI, which would bind to the periplasmic domain of T and prevent it from making holes. The structure of free RI shows a unique fold, capable of undergoing a large conformational change and domain swapping, which facilitates the release of the holin from the complex upon lysis triggering[15].
8. gp24(vertex protein)
The capsid of bacteriophage T4 is composed of the major capsid protein gp23, and a minor capsid protein gp24. Two glutamate to alanine mutations (E89A,E90A) can improve the stability, crystallizability and crystal quality of gp24 without affecting the overall folding.
Rational modification of the protein surface to achieve crystallization appears promising for improving crystallization behavior and crystal diffracting qualities. Surface mutation proved to be a better method than reductive methylation for improving diffraction quality of the gp24 crystals[16].
6 References
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[11]Jia, Z., Ishihara, R., Nakajima, Y., Asakawa, S., & Kimura, M. (2007). Molecular characterization of T4‐type bacteriophages in a rice field. Environmental Microbiology, 9(4), 1091-1096.
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[13]Pant, K., Shokri, L., Karpel, R. L., Morrical, S. W., & Williams, M. C. (2008). Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY. Journal of molecular biology, 380(5), 799-811.
[14]Leiman, P. G., Shneider, M. M., Mesyanzhinov, V. V., & Rossmann, M. G. (2006). Evolution of bacteriophage tails: Structure of T4 gene product 10. Journal of molecular biology, 358(3), 912-921.
[15]Krieger, I. V., Kuznetsov, V., Chang, J. Y., Zhang, J., Moussa, S. H., Young, R. F., & Sacchettini, J. C. (2020). The Structural Basis of T4 Phage Lysis Control: DNA as the Signal for Lysis Inhibition. Journal of molecular biology, 432(16), 4623-4636.
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