1. Transcriptomic Characterization of Bacteriophage Samisiti12
Chloe Standridge, Emmanuel Adeyemo, Farshad Hamid, Swapan Bhuiyan, Lee E. Hughes
Department of Biological Sciences, College of Arts and Science
Introduction
Bacteriophages are a group of categorized viruses that infect a bacteria
host. There are approximately 1031
phages present in a variety of
environments on Earth. Their vastness allows phages to be profoundly
unique with unusual characteristics that set them apart from other
organisms. These viruses possess small genomes, and phages with a high
level of similarity within their genomes are grouped into clusters, and
furthermore categorized into sub-clusters. Phages have a lytic life cycle,
but under certain circumstances, phages may undergo an alternative
lysogenic life cycle that creates a lysogen. A transcriptomic analysis
reveals portions of the genome that are expressed at different stages of
the life cycle. Phage Samisti12 from sub-cluster BE1 will be placed in a
culture of Streptomyces griseus and a sample of the phage population will
be removed at various time points of the infection cycle. RNA sequencing
will be performed on the extracted samples and analyzed to reveal the
portions of the genome expressed at different stages of the infection
cycle. From this analysis, genes required early or late in the life cycle can
be identified.
Methods
Rohwer, Forest Youle, Merry, and Maughan, Heather. Life in Our Phage World.
Web. <https://http://2015phage.org/art.php > Wholon. December 2014.
Halleran A, Clamons S, Saha M (2015) Transcriptomic Characterization of an
Infection of Mycobacterium smegmatis by the Cluster A4 Mycobacteriophage
Kampy. PLoS ONE 10(10): e0141100. doi:10.1371/journal.pone.0141100
Bibliography
Hypotheses
Due to the fact that synteny of bacteriophage genomic content is highly
conserved in phage populations, sequences on the right arm of the genome
are transcribed early in the infection cycle and sequences on the left arm of
the genome are transcribed late in the infection cycle. Genomic content on
the right arm encodes for functional proteins that are involved with DNA
synthesis, metabolism, and host immunity repression. Genomic content on
the left arm encodes proteins necessary for virion construction. DNA
sequence repeats present within the genome of Samisiti12 indicate
structural proteins to be present early and late in the lifecycle.
1. A Multiplicity of Infection (agent:target) equivalent to 10:1 was calculated to determine volumes of S. Griseus
culture and Samisiti12 HTL required for successful host infection
2. Log phase culture of S. grisues was infected with Samisi12 HTL and placed in a shaking incubator at 30⁰C for 10
minutes to ensure phage absorption
3. Culture was centrifuged and pellets were washed with Phage Buffer to remove unabsorbed phage particles
4. Pellets were re-suspended in Nutrient Broth and placed in shaking incubator at 30⁰C for the remainder of the
experiment
5. Latent period experiment was performed to determine the time points to measure mRNA expression—titer
assay was evaluated for samples removed at the following time periods: 0 minutes, 30 minutes, 60 minutes, 90
minutes, 120 minutes, 150 minutes, 180 minutes
6. After latency experiment, samples were removed at 30 minutes, 60 minutes, and 120 minutes for RNA isolation
7. Prepare samples for sequencing—follow standard procedure as listed in the RNeasy RNA isolation kit; Convert
RNA to cDNA using SuperScript III Reverse Transcriptase: Evolution of the SuperScript Reverse Transcriptases kit
8. Samisti12 cDNA will be sequenced and analyzed to confirm early transcription of functional proteins involved
with DNA processes and late transcription of structural proteins
9. Sequenced genome will be analyzed to measure differences in mRNA expression throughout phage lifecycle
Figure 2: Phamerator map that indicates similarities between genomes of bacteriophage Jay2Jay and bacteriophage Samisiti12 after thorough annotation. Repeats within Samisit12 genome are apparent at the beginning and at the end of the genome. Structural gene transcription Is expected take place early in the
infection cycle and late in the infection cycle.
Figure 3: Electron
microscopy image of
Samisti12 phage.
Figure 4: Detailed structure of
bacteriophage model.
https://micro.magnet.fsu.edu/cells/viruses/images/bacteriophage.jpg
Latency Period Evaluation
Time Period
30 minutes
60 minutes
90 minutes
120 minutes
150 minutes
180 minutes
Highest amount of cell bursting