Lecture 2: Introduction to Plasmids Flashcards
What is a plasmid?
A plasmid is a small, circular, double-stranded DNA molecule that is distinct and separate from a cell’s chromosomal DNA.
What is special about the genes carried by plasmids?
Plasmids often carry genes that provide bacteria with genetic advantages, such as antibiotic resistance.
What is the disadvantage to having plasmids during reproduction?
When a bacterium replicates and divides, all the plasmids contained within the cell are copied such that each daughter cell receives a copy of the plasmids. There is a fitness cost to the presence of plasmids; replicating multiple plasmids has an energy cost to the cell.
What are the 4 key sites on a plasmid?
- Restriction Site
- Origin of Replication
- Selectable Marker
- Multiple Cloning Region
What are restriction sites?
Locations on a DNA molecule containing specific (4-8 base pairs in length) sequences of nucleotides, which are recognized by restriction enzymes.
What are restriction enzymes?
. Restriction enzymes/ endonucleases cleave DNA into fragments at or near specific recognition sites. Allow for the insertion or removal of genes in the plasmid.
What is the ‘Origin of Replication’?
A sequence of DNA at which replication is initiated on a chromosome, plasmid or virus. For small DNAs, including bacterial plasmids and small viruses, a single origin is enough.
What is the role of a selectable marker?
Used to enable identification of successful insertion of a gen into a plasmid.
What is a ‘Multiple Cloning Region’?
DNA region within a plasmid that contains multiple restriction enzyme cut sites, allowing the plasmid to be cut and cloned at multiple different sites.
What are the 3 stages of plasmid replication?
- Initiation
- Elongation
- Termination
What happens during the initiation stage of plasmid replication?
Introduction of a specific single strand break (called nicking) in the replication origin by a Rep protein, which is encoded by the plasmid.
Catalysed by a plasmid-encoded initiation protein that recognises plasmid-specific DNA sequences and determines the point from which replication starts.
What happens during the elongation stage of plasmid replication?
Addition of amino acids to the newly synthesised strand by DNA polymerase III holoenzyme.
What happens during the termination stage of plasmid replication?
Nicking and re-joining of the displaced single-strand at the termination site.
What is a replisome?
A large protein complex that facilitates DNA replication, starting at the replication origin.
How are replisomes adapted to their function?
Replisomes contain several enzyme activities, such as helicases, primases and DNA polymerase. Replisomes form a replication fork to duplicate both the leading and lagging strand.
Why is control of replication and plasmid duplication needed?
To maintain a fixed concentration of plasmid molecule in the growing bacterial population. High levels of plasmid in the bacterial population has an energy cost associated.
How is plasmid duplication controlled?
RNA (antisense RNA), DNA sequences (iterons), or antisense RNA and proteins acting in concert, can be used to control plasmid replication.
How do restriction enzymes protect bacterial cells?
Restriction enzymes are thought to protect bacteria cells as phage and bacteria endonucleases have different degrees of methylation; this difference in methylation flags the phage and gives the bacteria the opportunity to destroy the phage.
How does methylation of the bacterial chromosome protect it from phage attack?
Methylation of bacterial chromosomal DNA prevents the activity of viral endonucleases. Plasmid cannot undergo methylation so it is the plasmid that is attacked during infection.
What are endonucleases?
Endonucleases are proteins that cleave the phosphodiester bond within the DNA.
What are exonucleases?
Exonucleases are proteins that cleave the phosphodiester bond at the terminal points of the DNA.
What is the role of endonucleases?
Endonucleases catalyse the hydrolysis of ester linkages within nucleic acids by creating internal breaks. Endonuclease rely on specific recognition sequences (restriction sites) in order to cut the DNA molecule.
How can endonucleases be used in plasmid engineering?
Endonucleases form sticky end breaks, depending on the endonuclease used, and can be used in plasmid engineering; the same endonuclease in needed to cut the desired gene, to allow for insertion into the plasmid.
What are the 2 responses to phage infection of bacteria?
- The bacterial endonucleases will destroy (inactivate) the invading viral DNA, thus preventing phage replication.
- The viral (phage) endonuclease will bind to the host DNA and destroy it.
