How to Read a Plasmid Map: pBR322 Quiz and Flashcards

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?

What is the consequence of having two plasmids with different antibiotic resistance genes, but the same replicon, in one cell?

What is the size of the linearized pBR322 plasmid?

What is the purpose of sequencing a plasmid via PCR after linearizing it with a restriction enzyme?

What do the black arrows on a plasmid map indicate?

What should you avoid changing in a plasmid?

What is a consequence of having plasmids with the same origin of replication?

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

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.