Bacterial Genetics Flashcards
What are auxotrophic bacteria?
Bacteria that cannot produce their own amino acids
How is complementation used to find if a certain gene is important?
Each gene is cloned into an expression plasmid and if the gene is important, it will return to wild type as a plasmid
How is reverse genetics done?
Reverse genetics links genes to biological function.
1. Mutating and over-expressing the gene
2. Using RNA-seq to determine changes in the transcriptome
3. Determining the phenotype on an array
How can differences in phenotypes be found?
BioLOG phenotype array which grows samples on different carbon sources to find defects
How are mutants used to determine genetics
- Identify genes involved in a particular function
- Be used to determine metabolic pathways and regulation, e.g with transcription factors
- Identifying sites of action for antibiotics
- Find conditional lethal mutants
- Help locate genes
What is complementation important in genetics?
Relationship between two different strains of an organism which have homozygous recessive mutants producing the same phenotype. Important genes can return to its own wild type
Why are mutants important in genetics?
- Help identify genes in a function
- Mutant phenotypes can inform about certain pathways
- Help understand metabolic regulation, e.g transcription factors
- Identifying antibiotic targets
- Conditional lethal mutants for isolating genotypes
- Locating genes
What is a transversion?
A single nucleotide polymorphism (SNP) between a purine and pyridamine
What is a base transition?
A single nucleotide polymorphism (SNP) between pyridamines or purines.
What types of mutations are there?
Substitutions
Insertions
Deletions
Inversions
Reversions
What is a nonsense mutation?
When a mutation changes a codon to a stop codon
What does slip strand mis-pairing do?
Turns specific proteins on or off. Done by pathogenic bacteria for immune evasion by switching expression of surface proteins (phase variation)
In what process does cytosine convert to uracil?
Deamination
Name some chemical sources of mutations
Base analogues, such as caffine
Base-modifying chemicals
Intercalaters which insert between bases
Deaminating/alkylating agents
Name a biological source of mutation
Transposons
Outline methyl mismatch repair
- MutS binds to the damage and recruits MutL to bind to the hemi-methylated site
- MutL recruits MutH
- This nicks hemi-methylated DNA
- UvrD unwinds DNA and exonucleases cut it off
- Polymerase I and ligase seal the strand
How are thymine dimers repaired?
- UvrA and UvrB are activated with ATP and bind to the site of error which forms a kink in DNA
- UvrB recruits UvrC (nuclease) which nicks the DNA
- UvrD helicase II unwinds DNA and Pol I and ligase seal the gaps
Outline base excision repair
- DNA glycosylase cuts out a faulty base, creating the AP site.
- AP endonuclease makes a cut in DNA which gets nicked further down
- Polymerase I and ligase seal the nick
How does recombinational repair work?
- When there is a gap in the thymine dimer preventing the strand being copied, RecA binds to ssDNA and lines up a homologous region
- This copies across a strand (strand invasion)
- The strand with the thymine dimer can be fixed with base excision. The other strand is sealed with polymerase and ligase.
When other DNA repair systems fail, what does error-prone repair do?
LexA (a repressor) is inactivated by DNA damage, so RecA activates a co-protease which helps with on the LexA repressor
sulA stops cell division
umuDC is an error-prone polymerase
UvrA is activated
What are RecA’s functions?
Recognising and binding ssDNA
Scanning for homology
Lining up chromosomes
What does Exo do in lambda red?
Exonuclease which degrades 5’ ends of the PCR product to give sticky ends
What does Beta do in lambda red?
binds to 3’ overhangs created by Exo and promotes annealing of cDNA (like RecA)
what does Gam do in lambda red?
binds to host RecBCD to inhibit Exo activity
What is arabinose?
Inducible promotor in the lambda red system which controls Exo, Beta and Gam genes. This is sensed by the transcription factor AraC
Outline homologous recombination
- RecBCD enters the end of DNA and unwinds it. It nicks DNA at a chi site and continues unwinding the DNA
- RecA assembles on ssDNA and scans dsDNA.
- This catalyses strand invasion and D-loop formation
- RuvAB assembles at the crossover point and pulls strands apart (branch migration)
- Endonucleases cleave one end of the D-loop, forming a holiday junction
- RuvC cleaves the holiday junction and broken ends ligate, completing the crossover
How are holiday junctions cleaved if donor and recipient DNA are circular?
A cointegrate forms and is cleaved by RuvC
How are holiday junctions cleaved if donor DNA is linear and recipient DNA is circular?
A second crossover is needed to maintain circularity
Outline gene doctoring
- In the presence of sucrose, SacB on the host plasmid produces levens which are toxic to bacteria
- I-SceI cleaves the plasmid
- λ red proteins recombine the linear plasmid and chromosome
- Cells which have lost SacB still have kanamycin resistance so grow in kanamycin
What are composite transposons?
Transposons containing 2 IS element, an antibiotic gene and catabolic genes
What are complex transposons?
Transposons containing the IS elements and additional genes.
E.g Tn3 has the res gene which encodes unlinks the cointegrate. It has tnpA which acts on inverted terminal repeats
What are conjugative transposons?
Transposons which transfer from one cell to another via conjugation. Non-autonomous so insert into plasmids or chromosomes
Outline non-replicative transposition
- Transposase is synthesised and binds inverted repeats
- This makes a staggered cut in target DNA
- IS elements attach to the cut ends
- The gaps are filled in, creating more duplicated regions
Outline the mechanism of a transposon being cleaved out of a gene
- Transposase aligns inverted repeats and flanking DNA
- A phosphodiester bond is cleaved on each strand at 3’ ends of both strands
- The 3’ OHs attack the intact ends, producing a hairpin structure.
- This moves to another DNA site and is re-nicked
What is TraDIS?
Transposons disrupt genes, inactivating them. If the disrupted gene is essential, the mutant doesn’t survive
Repeats flanking the transposon are read out with sequencing primers to recognise the position of transcription.
Outline transposon-mediated differential hybridisation (TMDH)
- Modified transposons with outward facing promotors are inserted into non-essential genes, inactivating them.
- Mutants are put together and lysed open
- The DNA is then digested into 60bp fragments and labelled with dNTPs.
- The T7 promotor is labelled red and SP6 green,
- Run-offs are hybridised onto a microarray and gibe a series of red and green RNAs, showing where the genome has inserted.
What sort of reporter gene fusions are used for known genes?
single or multi-copy reporter fusions
What sort of reporter gene fusions are used for unknown genes?
Modified transposons or promotor traps
How is LacZ used as a reporter gene?
Cumulative promotor activity can be measured with β-galactosidase in a colourmetric assay
How is cat used as a reporter gene?
cat encodes chloramphenicol which is an antibiotic, so is used to show resistant bacteria
How is lux used as a reporter gene?
lux encodes luciferase which is unstable and breaks down, allowing real time measurements
How is gfp used as a reporter gene?
gfp encodes green fluorescent protein
How do transcriptional fusions work?
Use promotors of the test gene and translational elements of the reporter gene to find transcriptional activity
How do translational fusions work?
Use promotors and translational elements of the test gene to find transcriptional and translational activity.
RNA polymerase binds at the open reading frame
Outline single copy reporter fusions
- The promotor of interest (POI) is ligated into a vector with a promotorless GPF gene
- The POI inserts at the multiple cloning site (mcs)
- The plasmid is electroporated into bacteria and inserts into the bacterial genome at attB
- Lipase gene geh is disrupted, so loss of lipase activity can be screened on egg yolk agar plates
Outline the GFP promotor trap
- FACS (fluorescent cell sorting) is used to separate cells by green/not green using a laser to find scattering of light
- Clones with active promotors (green) are removed
- Inactive promotors are put into an infection model
- Bacteria are then recovered and separated by FACS
What do crispr loci comprise of?
30bp repeats separated by 20-72 bp spacers
What are the 3 stages of CRISPR-Cas?
Adaptation: insertion of new spacers into the CRISPR locus
Expression: transcription of the CRISPR locus
Interference: degradation of mobile genetic elements
Outline the adaptation stage of CRISPR
- Phage DNA is cleaved and a spacer is incorporated into the host near a PAM site
- Cas proteins (Cas1, Cas2 and csn2) bind at the PAM site and cut up invading phage DNA
Outline the expression stage of CRISPR
- tracrRNA recognises repeat sequences and binds Cas9 nuclease
- RNaseIII digests bacterial DNA into spacers with 1 repeat and Cas9
- When Cas9 recognises the PAM site, it is activated and target DNA is cleaved
- RuvC cuts the top strand, HNH cuts the bottom strand. DNA is cleaved in two
- A linker loop is formed between 3’ crDNA and 5’ tracrRNA
- Repeat and tracrRNA are expressed
