Studying Virulence Factors 2 Flashcards
signature tagged mutagenesis (2)
- negative selection for virulence gene identification
- involves generating strains using Tn libraries
how does signature tagged mutagenesis work (5)
- mutate gene with a Tn carrying a unique tag
- introduce Tn mutants into chosen disease model
- harvest bacteria after certain amount of time
- use hybridization to identify tags
- isolate genomic material from clone and use Sanger’s sequencing to find which gene the transposon was inserted into
signature tagged mutagenesis: what is the purpose of looking for identity tags in the last step (2)
- looking for tags that were present in initial inoculum that are absent in recovered fraction
- identifies mutants that did not grow as they were mutated for a gene that represents a potential virulence factor
what are the advantages of signature tagged mutagenesis (2)
- can look at many mutants at a time using a few animals
- can identify gene important for growth and survival directly in vivo
signature tagged mutagenesis: disadvantage (2)
- usual Tn issues
- polar effect and insertion bias
polar effect (2)
- when a Tn is inserted into DNA that in transcripted into a polycistronic mRNA
- describes how the insertion may remove more genes that expected
how can we test for the polar effect (2)
- do complementary experiments to restore function
- do targeted deletions in each gene
Tn Seq
- looks for genes important to survival by comparing two conditions
what are some required elements for Tn Seq (2)
- high coverage of the Tn insertion sites; need to create a saturating library to determine all non-important genes
- use of the Himar Mariner Tn
Himar Mariner Tn (3)
- contains an antibiotic resistance marker
- contain MmeI restriction enzyme recognition sites within the Tn, but cut sites occur 20bp away from site (outside of Tn)
- MmeI leaves two-bp overhang for adaptor ligation
Tn Seq steps (5)
- mutanagize bacteria with Himar Mariner Tn several times to cover all possible genes
- digest chromosomal bacteria with MmeI
- ligate adaptors (MmeI cut site and Tn) for Illumina Sequencing
- compare read counts for each insertion site under different conditions
- identify loci required for growth under different conditions
IVET (2)
- in vivo expression technology
- promoter trap for in vivo expressed genes
IVET: reporter gene (2)
- β-Galactosidase protein
- promoter-less lacZ gene
how can we used IVET to identify Salmonella virulence factors: starting components (2)
- Salmonella genomic DNA
- engineered plasmid
how can we used IVET to identify Salmonella virulence factors: salmonella genomic DNA processes
- DNA is partially digested by Sau3AI restriction enzyme
how can we used IVET to identify Salmonella virulence factors: engineered plasmid components (6)
- origin of replication
- mob gene
- lacZY
- purA
- Bgl II cut site
- bla
how can we used IVET to identify Salmonella virulence factors: origin of replication
- it functions in E. coli, not in Salmonella
how can we used IVET to identify Salmonella virulence factors: mob gene
- required to mobilize plasmid for transfer to different cells
how can we used IVET to identify Salmonella virulence factors: lacZY
- promoter-less reporter construct
how can we used IVET to identify Salmonella virulence factors: purA
- promoter-less gene required for purine synthesis
how can we used IVET to identify Salmonella virulence factors: Bgl II (2)
- linearizes plasmid
- compatible with Sau3AI cuts
how can we used IVET to identify Salmonella virulence factors: experimental set-up (2)
- use a model system
- find avirulent strain that does not infect host model
how can we used IVET to identify Salmonella virulence factors: mouse model and avirulent bacterial strain
- use purine auxotroph Salmonella that does not infect mice
how can we used IVET to identify Salmonella virulence factors: purine auxotroph synthesis
- make a chromosomal deletion of purA gene in Salmonella
how can we used IVET to identify Salmonella virulence factors: first 1/2 of steps (purA importance) (5)
- ligate Sau3AI-digested Salmonella DNA and Bgl II-digested pIVET plasmid into E. coli
- activate mob gene to transfer into LoF purA Salmonella via conjugation
- pIVET will integrate into Salmonella purA auxotroph via homologous recombination, creating a library of Salmonella clones
- pool library and inject into mice
- mash up spleen and collect surviving bacteria (PurA+ phenotype) into a petri dish
how can we used IVET to identify Salmonella virulence factors: what are the characteristics of the surviving bacteria from the mouse spleen (2)
- fragments successfully recombined with a promoter
- gene with promoter turned on is thought to be necessary for survival in the mouse
how can we used IVET to identify Salmonella virulence factors: second 1/2 of steps (lacZ importance) (4)
- plate bacteria on media containing substrate for β-galactosidase (X-gal)
- if β-galactosidase is expressed, it will cleave X-gal and the colony will appear blue
- collect colonies that appear white and discard colonies that appear blue
- sequence insertion to find the gene that was expressed in white colonies
how can we used IVET to identify Salmonella virulence factors: importance of white colonies (3)
- β-galactosidase does not cleave X-gal; no blue appears
- these colonies are expressed in vivo, but not in vitro
- these are genes thought to be needed for survival specifically in the mouse model
how can we used IVET to identify Salmonella virulence factors: importance of blue colonies (2)
- β-galactosidase cleaves X-gal; blue appears
- these colonies are expressed in vivo and in vitro
how can we used IVET to identify Salmonella virulence factors: what should happen after the experiment
- LoF or biochemical experiment is needed to test the virulence/essential-ness of the gene
how can we used IVET to identify Salmonella virulence factors: why might genes that are only expressed in vivo have no effect on virulence (3)
- several genes may be under the same promoter
- there may be redundant factors (eg. 10 redundant adhesion factors); knocking out one does not affect the virulence as compensation by other factors will occur
- gene may be involved in house-keeping functions
IVET: disadvantages (2)
- genes expressed in vivo are not confirmed to be virulence factors; subsequent testing must be done
- IVET does not tell us where or when the genes were turned on
DFI (3)
- differential fluorescence induction
- promoter trap technology
- uses fluorescence-based selection for intracellularly-induced genes
DFI: reporter construct (2)
- promoterless gfp gene
- green fluorescence proteins
DFI steps: rough collection (6)
- make library of random fragments of genomic DNA
- insert upstream of the promoterless gfp gene in a plasmid
- infect host cell (eg. macrophage) with plasmod
- sort and collect fluorescing macrophages
- lyse macrophages and plate bacteria on lab media
- sort and collect bacteria (from lab media) with lowest fluorescence
DFI steps: refined collection/observation (5)
- use collected bacteria to infect macrophages again
- see enrichment of fluorescing macrophages
- lyse macrophages and collect bacteria
- study each bacterium in greater detail, paying attention to timing, levels and location of expression
- sequence cloned fragments and identify the gene
hybridization-based techniques (3)
- used for gene expression studies
- used to compare the relatedness of 2 bacterial strains
- involves dsDNA -> ssDNA
gene expression experiments (2)
- exposure of virulent strains to two different conditions
- compare virulent vs avirulent strains
subtractive hybridization
- used to identify gene sequences found in virulent strains, but not avirulent ones
subtractive hybridization steps (6)
- label DNA from “avirulent” strain with biotin (after adding linkers)
- combine all the fragments from the virulent and avirulent strain
- denature and reanneal DNA
- use streptavidin to remove biotinylated DNA
- what’s left (fragments without streptavidin) are fragments unique to the virulent strain
- clone fragment into plasmid and sequence the fragment
subtractive hybridization: denature and reanneal steps (3)
- mix avirulent + virulent DNA
- avirulent + biotin strands in excess
- complementary strands will hybridize: all virulent fragments, avirulent fragments, and shared virulent/avirulent strands will hybridize
how can we use subtractive hybridization for gene expression experiments (4)
- one strain (virulent) exposed to two different conditions
- collect mRNA
- convert to cDNA; reverse transcriptase and random primers
- do subtractive hybridization experiment
DNA microarrays
- used for gene expression studies or strain (DNA) comparisons
microarray (2)
- miniaturized device containing short single-stranded DNA oligonucleotide probes attached to solid substrate
- $100 per chip
microarray probes (2)
- designed to have sequences complementary to segments of one or more target organism genomes
- 20,000 probes
microarray probe synthesis (4)
- mechanically spotted
- sprayed
- using an ink jet print head
- synthesized using photochemical reactions
gene expression using microarrays: steps (7)
- expose virulent strain to 2 different conditions
- collect mRNA from each conditions
- convert mRNA into cDNA
- label cDNA with fluorescent probes, using different fluorescence for each condition
- mix and use to hybridize to an array
- array has every gene from the organism
- compare expression patterns (fluorescence colours)
microarrays to compare 2 strains (2)
- collect DNA from strain A and use it to hybridize to array from strain B
- if there are spots that don’t fluoresce on array, the genes are missing from strain A
DNA microarray: disadvantages to comparing two strains
- won’t identify any unique genes from hybridized strain
pangenome array
- DNA microarray with genes from many, many bacteria
RNA Seq (2)
- used to detect and quantify mRNA levels
- will also find small (non coding) RNA (sRNA)
RNA Seq: steps (7)
- extract RNA
- deplete rRNA and tRNA
- fragment the RNA
- convert to cDNA
- add adaptors, amplify library, and do Illumina sequencing
- align sequences to genome using bioinformatics
- the number of sequence reads is roughly proportional to amount of mRNA for a particular gene
what does each cluster on an Illumina sequencing chip represent when doing RNA Seq (2)
- one mRNA transcript for gene X in the original sample
- a cluster is the bridge-amplified produce of a single transcript
how do we measure RNA expression using RNA seq (3)
- count up number of identical sequences from each cluster on the chip
- map sequences to genome
- compare experimental conditions
RNA seq: advantages (3)
- can use RNA seq to compare bacteria grown under different conditions
- may discover novel genes
- small non coding RNA and antisense RNA can be found
dual RNA seq (2)
- can be used to find in vivo expressed genes
- can sequence bacterial and host cell transcripts (cDNA) at the same time
dual RNA seq: steps (5)
- infect cells with fluorescently labeled bacteria
- sort for infected cells to get a homogenous starting population
- extract RNA, depleting rRNA and tRNA
- convert to cDNA and sequence
- align to host genome and the bacterial genome separately
protein/proteome microarrays: expression plasmid example
- 6xHis
- promoter
- terminator
- ribosome binding site
- V5 tag
protein/proteome microarrays: steps before array (5)
- start with bacterial genome
- amplify all open reading frames (genes)
- clone into an expression plasmid
- express the proteins using an in vitro transcription and translation system
- do not need to purify the proteins
protein/proteome microarray: steps after array (4)
- use array with nickel-NTA (Ni++) which binds 6xHis tags
- anti-V5 tag antibody confirms expression of the construct
- add serum from patients or from experimental model
- if antibody binds to protein, a signal will be produced
