MIMG 102 Lecture Notes - Lecture 5: Microbiological Culture, Dna Replication, Virus

• School: UCLA
• Department: Microbiology, Immunology, and Molecular Genetics
• Course: MIMG 102
• Professor: Dasgupta Asim

Document Summary

Overview: dsdna ~ 40kb, virion: complex, icosahedral head & short tail tail recognizes e coli receptor & binds, more completely dominates host cell than m13. Pulse-labeling of proteins in t7-infected e coli: when pulse labeling, use amino acids radioactively labeled -> incorporated into protein. E coli protein labeled at the beginning, together with viral protein. High moi: 5 20 pfu/cell, e. g. one-step growth, all cells infected. With 5-20 virus particles per cell, at least 1 will be active (proteins not denatured) & infect the cell. Low moi: 0. 01 pfu/cell e. g. 1 virus for 100 cells: procedure. Add t7 to bacterial culture at time = 0, infecting 99% cells (moi =5) Remove 5 ml aliquots every 2 min. immediately after removing the aliquot, add 35s-methionine for 30 sec to pulse- label proteins lyse cells in sds. Prepare the host cell for viral replication. Gene 0. 3 protein inactivates host cell restriction enzyme. Gene 1 protein -> t7 rna pol absolutely required.