BPS310_assignment6_2023

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University of Ottawa *

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3101

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Biology

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Jan 9, 2024

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BPS3101 Assignment 6 1. A graduate student studying the pathogenic bacteria Acinetobacter baumannii made cDNA from planktonic cells and biofilm cells and performed RNA-Seq on both samples. She aligned her sequencing reads to a locus of the A. baumannii genome as shown. a.) Which genes are on an operon together? Explain which data supports this? yfgC and yfgD and yfg E and yfgF. b.) What is the most expressed transcript from the locus in Planktonic culture? yfgA c.) Order the transcripts from largest increase in relative expression between biofilm and planktonic cells to the largest decrease in relative expression. yfgCD, yfgB, yfgEF, yfgA. d.) When this data was compared to microarray transcriptional profiling, the microarray data provided lower expression levels for the most highly expressed transcripts. Why did this occur? Saturation of the microarray data. This occurs when there is more labelled cDNA amplicons. in the sample than there are oligos on the slide 2) CRISPR/Cas9 can be used to create indels or replace a gene in the genome with a specific sequence. A) Draw a figure showing how this system can be used to cut double stranded DNA. Clearly label the double stranded gDNA to be cut and any biopolymers used for the DNA cleavage. Indicate what leads to the sequence selectivity of DNA cleavage.

BPS3101 Assignment 6 B) Identify the two mechanisms used for double stranded DNA repair. Which generates indels and which can be used to insert new DNA into the genome? 1-Indels – non homologous end joining (NHEJ) 2- Insert new DNA – homology directed repair (HDR) 3) Regular transposon mutagenesis was unable to generate a Bacillus subtilis mutant able to survive on the antibiotic fosfomycin but a transposon with an outward facing promoter (shown below) enabled one clone out of a ten thousand clone library to survive. a.) Design a primer based on the transposon sequences shown to be used with this arbitrary primer 5’CTTGAN3’ to amplify the gene impacted by this transposon. Note this strategy is called arbitrary PCR. 5’GTGCAA3’ b.) Assuming B. subtilis has 50% GC content, what is the expected maximum length of your arbitrary PCR product? ess than or up to around 1024 bp. c.) What sequencing technology would you use to obtain the sequence of this PCR product? Sanger d.) Is this “loss of function” or “gain of function” mutagenesis? Justify your answer. Gain of function. the outward facing prompter Turing the gene on 4) Define these terms and if it is a technique in genomics explain what it is used for. siRNA: siRNA is a class of double-stranded RNA molecules, usually they are 20-25 nucleotides long. They play a role in the regulation of gene expression siRNA is often used as a tool to selectively downregulate the expression of target genes. Paralog: are genes that have evolved from a common ancestral gene through gene duplication 5’ RACE: is a molecular biology technique used to amplify and sequence the 5' end of a specific mRNA transcript. It helps in finding the transcription start site of a gene and obtaining information the UTRs of mRNA. 5’TGCAAT3’

BPS3101 Assignment 6 Phage display: is a technique to study protein-protein, protein-peptide, and protein-ligand interactions involves the expression of peptides or proteins on the surface of bacteriophages and the subsequent selection of specific binding peptides or proteins. b ion: Fragment of a peptide generated in a mass spectrometer

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