Podcast Beta
Questions and Answers
What is the primary reason why two pBR323-derived vectors cannot be maintained in one cell, even if they have genes for different antibiotic resistances?
What is the consequence of cutting the plasmid at the PvuI restriction site?
What is the function of the rrnBT2 terminator in the pTLNX plasmid?
What is the primary function of the beta-lactamase enzyme?
Signup and view all the answers
What is the consequence of having two plasmids with different antibiotic resistance genes, but the same replicon, in one cell?
Signup and view all the answers
What is the size of the linearized pBR322 plasmid?
Signup and view all the answers
What is the purpose of sequencing a plasmid via PCR after linearizing it with a restriction enzyme?
Signup and view all the answers
What do the black arrows on a plasmid map indicate?
Signup and view all the answers
What should you avoid changing in a plasmid?
Signup and view all the answers
What is a consequence of having plasmids with the same origin of replication?
Signup and view all the answers
Study Notes
Plasmid Basics
- pBR322 has two antibiotic resistance genes: tet (tetracycline resistance) and amp (ampicillin resistance)
- These genes encode an efflux pump (tetR) and beta-lactamase (ampR) that excrete tetracycline and ampicillin from the cell, respectively
- beta-lactamase is not specific in degrading penicillin-derived antibiotics
Cloning Considerations
- When using restriction enzyme sites to clone a gene of interest, look carefully at which sites fall within the antibiotic resistance gene
- Disruption in a gene leads to inactivation of gene function, including antibiotic resistance
Plasmid Replication and Transcription
- Plasmids often include transcription promoters and terminators derived from E. coli phages
- The origin of plasmid replication (ori) is essential for cloning and should not be changed
- Plasmids with the same origin are often incompatible
- Plasmid maps show the direction of transcription with black arrows
Plasmid Incompatibility
- Defined as the inability of different plasmids to be maintained in one bacterial cell
- Occurs when multiple plasmids within one cell have the same replicon and/or partitioning system
- The replicon: plasmids with the same replicon are incompatible because they compete for the same replication control machinery within the cell
Reading Plasmid Maps
- Plasmid maps show the size of the linearized plasmid, restriction sites, and direction of transcription
- Always check the plasmid sequence before starting a new project
- Ensure restriction enzymes don't cut the insert, antibiotic resistance genes, or the ori
Studying That Suits You
Use AI to generate personalized quizzes and flashcards to suit your learning preferences.
Description
Learn to read and understand plasmid maps, starting with the classic pBR322 plasmid. Identify key features, restriction sites, and size of the linearized plasmid. Essential knowledge for molecular cloning and genetic engineering.
