Discipline: Biological Sciences
Subcategory: Cancer Research
Mashunda Longmire - Alabama State University
Co-Author(s): Deepa Bedi, Tuskegee University,Tuskegee, AL; Komal Vig, Alabama State University, Montgomery AL
Bacteriophage also known as ‘phage’ is a virus that infects a bacteria and reproduces inside it. Phage display is important in cell targeting and therapy. Phage display targets selected proteins on the surface of phage. The purpose of this study was to select phages from the phage library which will specifically bind to MDA-MB 231 cells and can bypass endosomes. Chloroquine phosphate is the endosomal inhibitor that was used to disrupt the endosome in the MDA-MB 231 breast cancer cells. A series of phage selections on MDA-MB 231 breast cancer cells were performed on cells with the endosomal inhibitor, chloroquine. Binding assays were performed to investigate if the selected phages could bind specifically to the MDA-MB 231 cells. Three rounds of phage selection were performed. In the first round phages were sequentially selected to plastic, to serum and to MDA-MB 231 cells to acquire cancer cells binding specific phages. Cells were washed with elution buffer to wash unbound phages followed by lysis buffer to break the cells to release selected phages. Phage tittering was performed on all washes, input, eluate and lysate using E.coli and plaque colonies were counted. The selected phages in lysate and eluate were amplified using E. coli. Amplified phages were centrifuged and supernatant containing phages was precipitated using PEG NaCl. These steps were repeated in second and third round of phage selection in MDA-MB 231 cells. Binding of the phage to the target MDA-MB231 cells in the third round were tested by phage ELISA using phage specific M13 antibody. ELISA results show 3.8 times higher binding to the MDA-MB231 cells compared to untreated cells. Phages will be further tested for their ability to escape endosomes.Not Submitted
Funder Acknowledgement(s): This work was supported by US Dept. of Education, The Minority Science and Engineering Improvement Program (MSEIP) (P120A150008) to Dr. Komal Vig (PD) and by NSF-CREST (HRD-1241701) to Dr. Shree R. Singh (PI).
Faculty Advisor: Komal Vig, email@example.com
Role: In this research project I conducted the actual experiment, calculated the results and drafted the abstract.