Gardener 6-10 Flashcards
What do agrobacterium cause crown galls to make?
Opines (e.g. octopine, nopaline, mannopine)
What do agrobacterium use opines for?
As a C and N source for growth
Do all agrobacterium cause crown galls?
No, avirulent strains cannot as they lack the Ti plasmid
What is transposon mutagenesis?
the movement of genes into a host’s chromosome
Explain mutant isolation experiment with E. coli and A. tumefaciens
E.Coli is auxotrophic and has suicide vector with a selectible marker (Tn5) which is conjugable.
A. tumefaciens is prototrophic and has a Ti plasmid.
Mating occurs. Isolate the agrobacterium with the suicide vector by plating on minimal media with kanamycin.
Those with vector will e unable to replicate as one daughter will not get vector and not be kanamycin resistant, so colony is not formed.
If vector has integrated into the Ti plasmid or the host chromosome colony will form.
Differentiate between these by mating experiments with an agrobacterium without Ti plasmid and select for growth with opines and for ability to form crown galls (to check virulence)
T-DNA includes what?
Tumour production and nopaline synthesis genes
CHow are vir genese sequenced?
Use primers and use inverse pPCR. Identify ORF and amplify. Express in plasmid vector.
Can also put Kan resistance in middle, and clone Kan and adjacent segments and then select via Kan
What are a few examples of Type Iv Secretion Systems?
Agrobacterium => DNA into plants
H. pylori => pick up DNA from outside, inject DNA into human
What is a Type IV secretion system?
h
How many times do you have to hit each gene for 99% coverage?
4,6 times. Much over this amount for ‘saturation’
How many membranes does a T4SS go through?
3 - IM, OM, Host M
