Download Bacterial genetics

Bacterial genetics
by
E. Börje Lindström
This learning object has been funded by the European Commissions FP6 BioMinE project
Definitions
• Gene:
a DNA sequence that determines
- the aa-sequence in a peptide/protein
- rRNA
- tRNA
- operator (O)
- promoter (P)
•
Allele:
- a different form of a gene (wt, mutant)
• Genotype:
- the total amount of genes in an organism
•
Phenotype:
- those properties that are expressed
Definitions, cont.
• Mutation:
- a permanent change in the genotype
- spontaneous (frequency 10-10—10-5)
- induced (frequency >10-5)
• Mutant:
- an organism that has a changed genotype
• Two types of mutants:
-Gain of a property
(possible to select for)
-Loss of a property
(not selectable)
How to find the mutant?
• Those who are selectable:
-e.g. Antibiotic sensitive  resistant
- streak out the culture on a plate containing
the antibiotic.
• Those who are not
selectable:
- e.g. Prototroph (wt)  auxotroph (mutant)
1)
Enrichment with the e.g. penicillinmethod
2)
Screening with ’replica plating’
Inoculum
Penicillin-method
- Only prototrophs can grow  are
killed
MM-medium +
pen
- Change the medium
-All bacteria will grow
- both prototrophs and auxotrophs
- the auxotrophs are enriched
Complex medium without
penicillin
Replica plating
Replica plating, cont.
Nomenclature
Model system: tryptophan
• Genotype:
- trp- (trp+); (A, B, etc.)
• Phenotype:
- Trp+; Trp-
• Amino
acid:
try
Transfer of DNA between
bacteria
Donor
Recipient
Three processes:
1) Transformation:
- f ree donor-DNA  recipient
2) Transduction:
- donor-DNA within a virus (Phage)  recipient
3) Conjugation:
- cell-to-cell contact between donor and
recipient
Transformation
Transformation, cont.
The Donor
The Recipient
- Cultivate, make
competent cells
- lyse and cut the
DNA into pieces
Free DNA (10
genes/ fragment)
ds DNA
- Uptake of
DNA (ds or
ss)
- homologue integration
- transformed cell
Transduction
Two types:
• General transduction
-Model system: P1- E. coli
- P1 a virulent phage
• Specific transduction
- Model system: phage l – E. coli
General Transduction
Specifik transduction
Specifik transduction, cont.
Conjugation
Three different bacteria:
F-plasmid
Integrated
Free
FRecipient
(female)
F+
pili
Hfr (high frequency
of recombination)
Donors (males)
Integration of the F-plasmid
IS-element
oriT
Cross no. 1
• F+ x F-
F-  F+ (all)
The F-plasmid is transferred:
- a ss-DNA is transferred
- new DNA synthesis occur in both cells
- the complete F-plasmid is transferred
- rapid spread of the F-plasmid
Cross no. 1, cont.
Cross no. 1, cont.
Cross no. 2
• Hfr x F-
F-  F- (all)
Course of events:
1) Cell-to-cell contact
- pili promote a conjugation
bridge (Week)
2) ss-brake at oriT in Hfr
3) One of the Hfr-strands is transferred
– ori T first
4) Pairing of homologue DNA-segments
5) Integration and exchange of genetic material
6) Brake down of linear extra-chromosomal DNA
7) A conjugant/ recombinant has been constructed
-new synthesis in both cells
- constant transfer speed (takes 100
min for E. coli)
Cross no. 2, cont.
Cross no. 2, cont.
40)
A+ B+ C+
Ori T
A-
50)
Ori T
B-
- Partial diploid
C-
AA+ B+
B-
C-
C+
- Integration and exchange
Cross no. 2, cont.
Q: Why is streptomycin added to the
plate?
Summary
• Obtaining a recombinant/conjugant require:
- genes must have been transferred from a donor to a recipient
- exchange of genes must occur