Major
Biology
Anticipated Graduation Year
2021
Access Type
Open Access
Abstract
STN1 is a subunit of the CTC1-STN1-TEN1 (CST) protein complex. The CST complex is a highly conserved single-stranded DNA binding protein, that plays an important role in telomerase maintenance and promoting DNA replication under replication stress. Telomeres are regions at the end of the chromosomes that protect our DNA and maintain genome stability. Genome instability is one of the main causes of cancer and aging-related diseases. The activity of the STN1 component of the CST complex has been tied to rescuing genome stability under conditions of replication stress, and STN1 dysfunction has been associated with cancer and apoptosis. Therefore, further understanding of STN1 function is essential for studying cancer and chemotherapeutic agents. In this study, we cloned the STN1 gene and created a flag tag for STN1 to provide a tool for performing transient transfection and identifying STN1’s interacting partners. We successfully amplified STN1 with PCR, digested purified STN1 and pCL-Flag-myc-puro cloning vector with restriction enzymes, ligated the two DNAs, transformed competent Escherichia coli bacteria, and screened the plasmid for insertion. Our results from the PCR, restriction enzyme mapping, and DNA sequencing verified that we successfully cloned the gene, flag-STN1-flag, into a pCL-flag-myc-puro vector from pBabe-puro.
Faculty Mentors & Instructors
Dr. Emma Feeney, Department of Biology
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.
Subcloning of Flag-STN1-Flag from pBabe-puro to pCL-Flag-myc-puro to Test Relationship Between STN1 and Chemotherapeutic Agents
STN1 is a subunit of the CTC1-STN1-TEN1 (CST) protein complex. The CST complex is a highly conserved single-stranded DNA binding protein, that plays an important role in telomerase maintenance and promoting DNA replication under replication stress. Telomeres are regions at the end of the chromosomes that protect our DNA and maintain genome stability. Genome instability is one of the main causes of cancer and aging-related diseases. The activity of the STN1 component of the CST complex has been tied to rescuing genome stability under conditions of replication stress, and STN1 dysfunction has been associated with cancer and apoptosis. Therefore, further understanding of STN1 function is essential for studying cancer and chemotherapeutic agents. In this study, we cloned the STN1 gene and created a flag tag for STN1 to provide a tool for performing transient transfection and identifying STN1’s interacting partners. We successfully amplified STN1 with PCR, digested purified STN1 and pCL-Flag-myc-puro cloning vector with restriction enzymes, ligated the two DNAs, transformed competent Escherichia coli bacteria, and screened the plasmid for insertion. Our results from the PCR, restriction enzyme mapping, and DNA sequencing verified that we successfully cloned the gene, flag-STN1-flag, into a pCL-flag-myc-puro vector from pBabe-puro.