Plasmids and Conjugation Flashcards
(37 cards)
What is incompatibility?
The inability of plasmids to exist in the same cell if they have the same replication mechanisms
What are the three molecular forms that plasmids may take?
supercoiled, linear, open-circle
What is the copy number and how does the copy number of a plasmid affect partitioning?
Copy number is the number of plasmids per cell. Small high-copy number plasmids have random plasmid partitioning whereas large low-copy plasmids are directed into each daughter cell
What are the two types of host range in plasmids?
Narrow: plasmid will only replicate in related species
Broad: plasmid will replicate in many hosts
Give an example of a broad host range plasmid
RP4, can grow in gram -ve or gram +ve bacteria
What 5 characteristic may plasmids encode?
antibiotic resistance, metal(oid) resistance, virulence determinants, bacteriocin production (antimicrobial-kills bacteria without plasmid), biodegradative properties
What is a transposon (give an example)?
A DNA sequence with the ability to move to other parts of the genome e.g. bla (beta-lactamase), encodes ampicillin resistance
What 4 things would a useful vector plasmid need?
orin which replicates in host, antibiotic resistance (or B/W selection to determine uptake), multiple cloning sites, promoter (inducible/constitutive or strong/weak)
What are the advantages and disadvantages of a strong promoter?
adv: gene expression is consistent and high
dis: constitutive expression, not inducible, so not controllable
What makes a suicide vector and what is it purpose?
A suicide vector occurs if a plasmid has an oriV which cannot replicate in host of interest. Kills host if the host doesn’t take plasmid into it’s genome
How does a suicide vector work?
If a host cell takes up a plasmid but does not incorporate the plasmids DNA into it’s genome the plasmid will circularise and express genes which result in cell death
What is a shuttle vector and what is it used for?
A plasmid which propagates in two different species, can be used to clone a gene in one species and then transfer to another
What two origins of replication must conjugative plasmids contain?
oriV and oriT
Give three examples of conjugative systems (plasmids) with some extra detail
F plasmid: fertility factor, narrow host range
RP4: broad host range, pseudomonas
Ti plasmid: agrobacterium tumefaciens, vir and tra systems
What are the names of the sets of genes responsible for conjugal transfer between prokaryotes? What are the names of the sets of genes responsible for conjugal transfer between prokaryotes and eukaryotes?
Pro: tra/trb
Pro/Euk: vir
What are the 4 steps involved in conjugation?
- Mating pair formation (Mpf) - a pilus forms between 2 cells
- Retraction - pilus retracts bringing cells closer together,, plasmid strand nicked at oriT (by relaxase)
- DNA transfer (Dtr) - relaxase unwinds DNA & transfers strand through pilus, rolling circle replication replaces donor DNA strand
- Replication - complementary DNA strand synthesised in recipient
What is the role of the coupling protein in conjugation?
The coupling protein synchronises mating pair formation with DNA transfer and pumps the DNA across
What is the role of relaxosome? And where is it coded?
The relaxosome is a protein complex made up of relaxase and accessory proteins which facilitates plasmids during conjugation. Relaxase initiates conjugation by nicking the plasmid (at the nic site)
Where and which genes encode the relaxosome?
tra gene in the oriT
What are the 4 stages of the relaxosome formation?
- TraJ binds to inverted repeat closest to nic site
- TraI binds to TraJ-oriT complex (at nic site)
- TraK binds to bent region of oriT
- TraI-TraJ-oriT complex stabilised by TraH
What is another word for the sex pili? And what is it’s appearance (?
F pili,
What is another name for TraI?
Relaxase
During conjugation, in which order do the 4 Tra proteins bind?
TraJ –> TraI –> TraK –> TraH
How does the binding of TraI initiate conjugative transfer?
by cleaving a specific phosphodiester bond in oriT
