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MICB 308 > Studying Virulence Factors 2 > Flashcards

Studying Virulence Factors 2 Flashcards

1
Q

signature tagged mutagenesis (2)

A
  • negative selection for virulence gene identification
  • involves generating strains using Tn libraries
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2
Q

how does signature tagged mutagenesis work (5)

A
  1. mutate gene with a Tn carrying a unique tag
  2. introduce Tn mutants into chosen disease model
  3. harvest bacteria after certain amount of time
  4. use hybridization to identify tags
  5. isolate genomic material from clone and use Sanger’s sequencing to find which gene the transposon was inserted into
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3
Q

signature tagged mutagenesis: what is the purpose of looking for identity tags in the last step (2)

A
  • 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
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4
Q

what are the advantages of signature tagged mutagenesis (2)

A
  • can look at many mutants at a time using a few animals
  • can identify gene important for growth and survival directly in vivo
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5
Q

signature tagged mutagenesis: disadvantage (2)

A
  • usual Tn issues
  • polar effect and insertion bias
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6
Q

polar effect (2)

A
  • when a Tn is inserted into DNA that in transcripted into a polycistronic mRNA
  • describes how the insertion may remove more genes that expected
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7
Q

how can we test for the polar effect (2)

A
  • do complementary experiments to restore function
  • do targeted deletions in each gene
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8
Q

Tn Seq

A
  • looks for genes important to survival by comparing two conditions
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9
Q

what are some required elements for Tn Seq (2)

A
  • 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
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10
Q

Himar Mariner Tn (3)

A
  • 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
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11
Q

Tn Seq steps (5)

A
  1. mutanagize bacteria with Himar Mariner Tn several times to cover all possible genes
  2. digest chromosomal bacteria with MmeI
  3. ligate adaptors (MmeI cut site and Tn) for Illumina Sequencing
  4. compare read counts for each insertion site under different conditions
  5. identify loci required for growth under different conditions
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12
Q

IVET (2)

A
  • in vivo expression technology
  • promoter trap for in vivo expressed genes
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13
Q

IVET: reporter gene (2)

A
  • β-Galactosidase protein
  • promoter-less lacZ gene
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14
Q

how can we used IVET to identify Salmonella virulence factors: starting components (2)

A
  • Salmonella genomic DNA
  • engineered plasmid
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15
Q

how can we used IVET to identify Salmonella virulence factors: salmonella genomic DNA processes

A
  • DNA is partially digested by Sau3AI restriction enzyme
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16
Q

how can we used IVET to identify Salmonella virulence factors: engineered plasmid components (6)

A
  • origin of replication
  • mob gene
  • lacZY
  • purA
  • Bgl II cut site
  • bla
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17
Q

how can we used IVET to identify Salmonella virulence factors: origin of replication

A
  • it functions in E. coli, not in Salmonella
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18
Q

how can we used IVET to identify Salmonella virulence factors: mob gene

A
  • required to mobilize plasmid for transfer to different cells
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19
Q

how can we used IVET to identify Salmonella virulence factors: lacZY

A
  • promoter-less reporter construct
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20
Q

how can we used IVET to identify Salmonella virulence factors: purA

A
  • promoter-less gene required for purine synthesis
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21
Q

how can we used IVET to identify Salmonella virulence factors: Bgl II (2)

A
  • linearizes plasmid
  • compatible with Sau3AI cuts
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22
Q

how can we used IVET to identify Salmonella virulence factors: experimental set-up (2)

A
  • use a model system
  • find avirulent strain that does not infect host model
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23
Q

how can we used IVET to identify Salmonella virulence factors: mouse model and avirulent bacterial strain

A
  • use purine auxotroph Salmonella that does not infect mice
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24
Q

how can we used IVET to identify Salmonella virulence factors: purine auxotroph synthesis

A
  • make a chromosomal deletion of purA gene in Salmonella
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25
Q

how can we used IVET to identify Salmonella virulence factors: first 1/2 of steps (purA importance) (5)

A
  1. ligate Sau3AI-digested Salmonella DNA and Bgl II-digested pIVET plasmid into E. coli
  2. activate mob gene to transfer into LoF purA Salmonella via conjugation
  3. pIVET will integrate into Salmonella purA auxotroph via homologous recombination, creating a library of Salmonella clones
  4. pool library and inject into mice
  5. mash up spleen and collect surviving bacteria (PurA+ phenotype) into a petri dish
26
Q

how can we used IVET to identify Salmonella virulence factors: what are the characteristics of the surviving bacteria from the mouse spleen (2)

A
  • fragments successfully recombined with a promoter
  • gene with promoter turned on is thought to be necessary for survival in the mouse
27
Q

how can we used IVET to identify Salmonella virulence factors: second 1/2 of steps (lacZ importance) (4)

A
  1. plate bacteria on media containing substrate for β-galactosidase (X-gal)
  2. if β-galactosidase is expressed, it will cleave X-gal and the colony will appear blue
  3. collect colonies that appear white and discard colonies that appear blue
  4. sequence insertion to find the gene that was expressed in white colonies
28
Q

how can we used IVET to identify Salmonella virulence factors: importance of white colonies (3)

A
  • 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
29
Q

how can we used IVET to identify Salmonella virulence factors: importance of blue colonies (2)

A
  • β-galactosidase cleaves X-gal; blue appears
  • these colonies are expressed in vivo and in vitro
30
Q

how can we used IVET to identify Salmonella virulence factors: what should happen after the experiment

A
  • LoF or biochemical experiment is needed to test the virulence/essential-ness of the gene
31
Q

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)

A
  • 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
32
Q

IVET: disadvantages (2)

A
  • 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
33
Q

DFI (3)

A
  • differential fluorescence induction
  • promoter trap technology
  • uses fluorescence-based selection for intracellularly-induced genes
34
Q

DFI: reporter construct (2)

A
  • promoterless gfp gene
  • green fluorescence proteins
35
Q

DFI steps: rough collection (6)

A
  1. make library of random fragments of genomic DNA
  2. insert upstream of the promoterless gfp gene in a plasmid
  3. infect host cell (eg. macrophage) with plasmod
  4. sort and collect fluorescing macrophages
  5. lyse macrophages and plate bacteria on lab media
  6. sort and collect bacteria (from lab media) with lowest fluorescence
36
Q

DFI steps: refined collection/observation (5)

A
  1. use collected bacteria to infect macrophages again
  2. see enrichment of fluorescing macrophages
  3. lyse macrophages and collect bacteria
  4. study each bacterium in greater detail, paying attention to timing, levels and location of expression
  5. sequence cloned fragments and identify the gene
37
Q

hybridization-based techniques (3)

A
  • used for gene expression studies
  • used to compare the relatedness of 2 bacterial strains
  • involves dsDNA -> ssDNA
38
Q

gene expression experiments (2)

A
  • exposure of virulent strains to two different conditions
  • compare virulent vs avirulent strains
39
Q

subtractive hybridization

A
  • used to identify gene sequences found in virulent strains, but not avirulent ones
40
Q

subtractive hybridization steps (6)

A
  1. label DNA from “avirulent” strain with biotin (after adding linkers)
  2. combine all the fragments from the virulent and avirulent strain
  3. denature and reanneal DNA
  4. use streptavidin to remove biotinylated DNA
  5. what’s left (fragments without streptavidin) are fragments unique to the virulent strain
  6. clone fragment into plasmid and sequence the fragment
41
Q

subtractive hybridization: denature and reanneal steps (3)

A
  1. mix avirulent + virulent DNA
  2. avirulent + biotin strands in excess
  3. complementary strands will hybridize: all virulent fragments, avirulent fragments, and shared virulent/avirulent strands will hybridize
42
Q

how can we use subtractive hybridization for gene expression experiments (4)

A
  • one strain (virulent) exposed to two different conditions
  • collect mRNA
  • convert to cDNA; reverse transcriptase and random primers
  • do subtractive hybridization experiment
43
Q

DNA microarrays

A
  • used for gene expression studies or strain (DNA) comparisons
44
Q

microarray (2)

A
  • miniaturized device containing short single-stranded DNA oligonucleotide probes attached to solid substrate
  • $100 per chip
45
Q

microarray probes (2)

A
  • designed to have sequences complementary to segments of one or more target organism genomes
  • 20,000 probes
46
Q

microarray probe synthesis (4)

A
  • mechanically spotted
  • sprayed
  • using an ink jet print head
  • synthesized using photochemical reactions
47
Q

gene expression using microarrays: steps (7)

A
  1. expose virulent strain to 2 different conditions
  2. collect mRNA from each conditions
  3. convert mRNA into cDNA
  4. label cDNA with fluorescent probes, using different fluorescence for each condition
  5. mix and use to hybridize to an array
  6. array has every gene from the organism
  7. compare expression patterns (fluorescence colours)
48
Q

microarrays to compare 2 strains (2)

A
  • 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
49
Q

DNA microarray: disadvantages to comparing two strains

A
  • won’t identify any unique genes from hybridized strain
50
Q

pangenome array

A
  • DNA microarray with genes from many, many bacteria
51
Q

RNA Seq (2)

A
  • used to detect and quantify mRNA levels
  • will also find small (non coding) RNA (sRNA)
52
Q

RNA Seq: steps (7)

A
  1. extract RNA
  2. deplete rRNA and tRNA
  3. fragment the RNA
  4. convert to cDNA
  5. add adaptors, amplify library, and do Illumina sequencing
  6. align sequences to genome using bioinformatics
  7. the number of sequence reads is roughly proportional to amount of mRNA for a particular gene
53
Q

what does each cluster on an Illumina sequencing chip represent when doing RNA Seq (2)

A
  • one mRNA transcript for gene X in the original sample
  • a cluster is the bridge-amplified produce of a single transcript
54
Q

how do we measure RNA expression using RNA seq (3)

A
  1. count up number of identical sequences from each cluster on the chip
  2. map sequences to genome
  3. compare experimental conditions
55
Q

RNA seq: advantages (3)

A
  • 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
56
Q

dual RNA seq (2)

A
  • can be used to find in vivo expressed genes
  • can sequence bacterial and host cell transcripts (cDNA) at the same time
57
Q

dual RNA seq: steps (5)

A
  1. infect cells with fluorescently labeled bacteria
  2. sort for infected cells to get a homogenous starting population
  3. extract RNA, depleting rRNA and tRNA
  4. convert to cDNA and sequence
  5. align to host genome and the bacterial genome separately
58
Q

protein/proteome microarrays: expression plasmid example

A
  • 6xHis
  • promoter
  • terminator
  • ribosome binding site
  • V5 tag
59
Q

protein/proteome microarrays: steps before array (5)

A
  1. start with bacterial genome
  2. amplify all open reading frames (genes)
  3. clone into an expression plasmid
  4. express the proteins using an in vitro transcription and translation system
  5. do not need to purify the proteins
60
Q

protein/proteome microarray: steps after array (4)

A
  1. use array with nickel-NTA (Ni++) which binds 6xHis tags
  2. anti-V5 tag antibody confirms expression of the construct
  3. add serum from patients or from experimental model
  4. if antibody binds to protein, a signal will be produced

MICB 308 (22 decks)

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