Overexpressing sRNA in Helicobacter pylori with Cloning and Transformation
Faculty Mentor
Andrea Castillo
Document Type
Poster
Start Date
10-5-2023 9:00 AM
End Date
10-5-2023 10:45 AM
Location
PUB NCR
Department
Biology
Abstract
The bacterial stomach pathogen Helicobacter pylori infects half the global population and causes symptomatic diseases, like stomach ulcers and gastric cancer, in 10-15% of those infected. Helicobacter pylori uses variable gene expression to adapt to its hostile and ever-changing environment using small RNAs (sRNAs), which are short, noncoding sequences that regulate other RNAs. The cytotoxin-associated gene pathogenicity island (cagPAI) region of the genome is associated with greater severity of symptoms. Our goal is to learn if virulence is regulated by two sRNAs from the cagPAI region, Hpnc2620 and Hpnc2540, by comparing RNA gene expression in strains overexpressing (making an overabundance of) these sRNAs to the parent strain. We will create overexpression strains with cloning and transformation techniques. The sRNA genes will be introduced to bacteria using a plasmid vector, a small circular DNA molecule capable of carrying the gene insert, in a process called transformation. Genes associated with the plasmid will be expressed at high levels in the bacteria. We successfully transformed an alternate plasmid pTM117 into H. pylori and demonstrated a protocol for future transformations with these bacteria. Current work with plasmid pCTtnpR1 and sRNA genes includes polymerase chain reactions (PCR), agarose gel electrophoresis, and purifications to amplify, verify, and build the experimental plasmids. Helicobacter pylori is naturally capable of double crossover homologous recombination, which uses overlapping complementary ends to switch gene segments and will incorporate sRNA genes from assembled plasmids into chromosomal DNA. This work will allow future investigation of sRNAs, Hpnc2620 and Hpnc2540, for their RNA regulation.
Recommended Citation
Morgan, Olivia; McPeck, RN; and Castillo,, AR PhD, "Overexpressing sRNA in Helicobacter pylori with Cloning and Transformation" (2023). 2023 Symposium. 24.
https://dc.ewu.edu/srcw_2023/res_2023/p1_2023/24
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Overexpressing sRNA in Helicobacter pylori with Cloning and Transformation
PUB NCR
The bacterial stomach pathogen Helicobacter pylori infects half the global population and causes symptomatic diseases, like stomach ulcers and gastric cancer, in 10-15% of those infected. Helicobacter pylori uses variable gene expression to adapt to its hostile and ever-changing environment using small RNAs (sRNAs), which are short, noncoding sequences that regulate other RNAs. The cytotoxin-associated gene pathogenicity island (cagPAI) region of the genome is associated with greater severity of symptoms. Our goal is to learn if virulence is regulated by two sRNAs from the cagPAI region, Hpnc2620 and Hpnc2540, by comparing RNA gene expression in strains overexpressing (making an overabundance of) these sRNAs to the parent strain. We will create overexpression strains with cloning and transformation techniques. The sRNA genes will be introduced to bacteria using a plasmid vector, a small circular DNA molecule capable of carrying the gene insert, in a process called transformation. Genes associated with the plasmid will be expressed at high levels in the bacteria. We successfully transformed an alternate plasmid pTM117 into H. pylori and demonstrated a protocol for future transformations with these bacteria. Current work with plasmid pCTtnpR1 and sRNA genes includes polymerase chain reactions (PCR), agarose gel electrophoresis, and purifications to amplify, verify, and build the experimental plasmids. Helicobacter pylori is naturally capable of double crossover homologous recombination, which uses overlapping complementary ends to switch gene segments and will incorporate sRNA genes from assembled plasmids into chromosomal DNA. This work will allow future investigation of sRNAs, Hpnc2620 and Hpnc2540, for their RNA regulation.
