Lecture #4 -Bacterial Genetics Flashcards
Bacterial Gene Nomenclature
Bacterial genes and gene products are assigned names with 4 letters
- First three letters – refers to the function of the gene or the group of genes
- Fourth letter – Assigned alphabetically
Example - Fts genes = typically involoved in cell division –> knocking any of them out results in Filamentous temperatire-Sensative phenotype
- Gene family includes ftsA and ftsB etc.
Differentiating between bacterial genes and bacterial gene products
Genes – written in italics with the last letter capitilized
- Example – flsZ, blaB, dnaA
Proteins - same letters as the gene BUT they are written in regular typeface with the first and last letters capitilized
- Ex. FtsZ, BlaB, DnaA
Genetic loci - name of the gene followed by two colons (::)
- Example – if you were to insert the gene ftsA at the ftsZ locus you would write it as ftsZ::ftsA
Multiple genes or proteins in the same family can be written in shorthand
- Example – want to refer to both the ftsA and ftsB –> ftsAB
- Can do the same for proteins (both proteins - FtsAB)
Bacterial Genome (overall)
Often have 1 chromosome
Chromsome is usually circular (is lienar in a few species)
Chromosome has 1 ORI –> allows for bidrectional replication
There is a wide range of genome size across species
Bacteria range 500-8000 protein coding genes
Genes can be encoded on both strands
Bacetra has no introns
E.coli (overall)
E.coli - most commonly studied bacterial model organsim
- E.coli genome = 4.6 mega base pairs (Mbp) ; 100X fewer than humans
- E.coli genome has 4000 protein coding genes (5X fewer than humans)
Bacterial Transcripts
Bacteria expression elements (transcripts) can exist as a single gene (expression is driven by a promoter) –> Bacteria gene can be clustered into a Operon
Bacterial Operons
Operon genes are transcribed into 1 mRNA
- mRNA made = polycystronic –> mRNA is then translated = yields discrete proteins
- Bacteria gene can be clustered into a Operon
In operon – expression of a group of genes is controlled by 1 promoter
Genes in operon are usually functionally related
Function - Operon allows for the production of multiple proteins in the same pathway all at once
Transcription and Tranlsation in Bacteria
Transcription and translation in bacteria also differ for Eukaryotes:
1. Bacteria genes do not have introns – mRNA does not usually need to be spliced
- Expection = some bacteria have self splining introns but that is rare
2. Since there is no nucleus – bacterial mRNA does not need to be transported before translation= transcription and translation can occur simultaneous
Bacteria storage of genetic information
Bacetria = can contain a second type of storage system for genetic information in addition to the circular chrosomeome
Second class of DNA storage = plasmid
Plasmids
Plasmid = extra chromosomal indepentely replicated DNA found in bacteria
- Plasmids = usually circular + usuallly non-essential
Often have multiple copies of the same plasmid in 1 cell (common to have 1-100 copies per cell)
- Plasmids can be in different copy numbers
Plasmids = smaller than the bacterial chrosome (1-200 kb plasmid vs. 4.6 megabase genome)
Plasmids can either be incorporated into the genome or may exist as a seperate entity
Plasmids in nature
Many bacteria have naturally occurring plasmids
Natural plasmids in Bacteria can:
1. Confer Antibiotic resistnce
2. Increase virulence
3. Aide in metabolism of a substance
Plasmids in lab Use
Use:
1. Molecular cloning –> transferring a gene from a genome or plasmid into another plasmid
- Includes introduce a DNA sequence into Eukaryotic cells
2. Protein purification –> –> Plasmids can be used to produce and purify recombinant protein
3. Exogenous gene expression
- Used to study bacterial physiology and biological processes
4. Facilitate chromosome engineering
- Used to study bacterial physiology and biological processes
5. Introduce a DNA sequence into bacterial cells
Key features common to most plasmids
- Restriction enzymes sites
- 5’ and 3’ Primer Sites
- Promoter
- Antibiotoc resistnce gene
- Markers selectable by other means
- Example – gene that makes growth in the presence of sucrose toxic OR Gene that makes growth on a nutrient necessary - Origin of replication
Plasmids = Need all of the elements for gene expression (to have expression of the gene of interest)
- Example – need the promoter and the ribosome binding site
Plasmid Structure - Restriction enzymes sites
Purpose - Allows for insertion or excision of a gene of interest
- aka multiple cloning site (MCS)
Between the RE (MCS) have the gene of interest that we cloned in
- RE sites = surrounds the gene of interst
Plasmid Structure - 5’ and 3’ Primer Sites
Purpose - Allows for confirmation of gene insertion through PCR amplification and sanger sequencing
Location –> Primer sites = regions upstream and downstream of the inserted gene
Plasmid Structure - Promoter
Purpose - Allows gene to be expression
Location - Furtehr upstream than the primer sites
Types of promoters used:
1. Inducible promoter (used for protein expression)
2. Endogenous promoter that is activated by ceullar process or a constitutively active promoter
Plasmid Structure - Antibiotic resistance gene
Purpose - Allows for the uptake of the plasmid to be selected for by plating bacteria on media that contains the corresponding Antibiotic
- Function as a selectable marker –-> select for cells that have the plasmid
Plasmid Structure - Origin of replication
Purpose - allows the plasmid to be copied and maintained
ORI – determines the copy number of the plasmid in cells and the host range (broad vs. Narrow)
- Depending on how active the ORI is = affects the copy number
- Host range is based on the genes in the bacteria –> affect what types of bacteria can recognize the ORI and replicate/maintain the plasmid
- Broad host range = plasmid can be maintained in many species
High vs. Low copy number plasmids
Plasmids in high copy number are easily distributed to daughter cells through diffusion
Lower copy number plasmids can contain genes encoding for their own partitioning systems = ensures transmission to both daughter cells
Other things plasmids can contain
- Partitioning systems
- For plasmids in low copy number = need partition systmes (things that maintain the plasmid in the bacteria)
- ORIT (Origin of transfer) –> required for conjugation
- Reporter genes (Ex. LacZ)
- Genes for required replication
What is required for the production of the AB resistance gene product form the plasmid?
D –> promoter + ribosome binding site
- Promoter = drives transcription
- Ribosome binding site = loads the ribosomes to get the gene product to actually get AB resistance
DNA uptake and transfer Methods
- Binary Fission
- Transformation and Electorportion
- Conjugation
- Transduction
Binary Fission
Overall - Normal Cell Division
Most common mechanism of DNA transfer
Genome is replicated and identical copies are transfered to each daughter cell
Transformation (overall)
Transformation = uses electroporation or heat shock to get plasmid or DNA into cells (get bacteria to take up plasmids)
Purpose - Facilitate plasmid uptake into a cell
Process – shock the bacteria (either with heat or electricity) –> shock increases the permeability of the bacterial envelope –> increased permeability allows for the uptake of the plasmid –> after plasmid uptake the cells are plated on media containing antibiotic that the plasmid confers resistence to–> select for successful tranformants –> isolate colony/clone (clone will contain the plasmid) –> grow the clone in media that conatins the antibiotic
- Transformants = cells that have taken up the plasmids
- After isolating the clone/colony that has the plasmid –> grow the clone in media that contains the antibiotic to provide selective pressure for retaining the plasmid (IF grow in plain media then the clone could lose the plasmid )
What is needed for heat shock transformation
For heat shock transformation – need to treat the cells with salt prior to shocking the cells –> helps plasmids associated with the bacterial envelope = increases the frequency of plasmid uptake
- Example salt = Calcium chloride
Conjugation
Conjugation = allows for plasmid transfer between bacterial cells using naturally occuring gene transfer machinery to move the plasmid from 1 cell to another
Example – allows for transmission of the F plasmid among E.coli cells (plasmid has genes that promote its own replication and transfer between bacteria)
What do you need for conjugation to work
- Conjugation machinery in the donor cell
- Conjugation machinery = proteins that make that conjugative pillus
- Need enzymes that allow for nicking and transfer of plasmid DNA from donor to the recipient - OirT/bom on the plasmid –> recognized by the conjugation machinery
- Suceptible recipient
- Can test if the recipient can be targeted by the conjugation machinery of the donor
Conjugation Process
- To initiate conjugation – a pilus protrudes from the donor cell and adheres to the recipient cell –> the cells are brought together = cells now share cytosols
- Relaxasome makes a nick in teh F plasmid = allows each strand of the plasmid DNA to detach from one another
- One strand of the F plasmid is moved from the donor to the recipient cell and each strand of DNA is replicated in both cells
- Cells detach from each other
Survey of the organisms in the gut
Metagenomices shows us that make species are firmicutes (firmucutes are a large fraction of bacteria in the gut)
Activity #1 - culture bacteria and manipulate them to see how they contribute to the function of the microbiome
Goal = isolate new genetically tractable Firmicutes from human stool
Genetically tractable = Can take up and maintain a plasmid AND express the markers that are encoded on the plasmid
Approach – design a plasmid for conjugation from E.coli into a mixed population of organisms in human stool that would favor isolation of Firmicutes (Moving the plasmid from E.coli into a mixed popultion)
- Want to select for organisms that take up and maintain the plasmid
Plasmid will have - Firmicute derived ORI (PIM13)+ MCS (RE site)+ E. coli derived Ori (PMB1) + Antibiotic resistence gene + OriT
- Everything else required for conjugation is encoded in the E.coli donor chrosmes
Experiment done to isolate new genetically tractable Firmicutes from human stool
- Transform plasmid into E.coli
- Mix E. coli (door) with the stool sample to allow for conjugation(Plasmid to go from E.coli to bacteria in stool)
- Plate on selective meidua that will only allow succssfully conjugated Firmicutes to grow
- Plate on mediaum that allows us to isolate for bacteria that took up the plasmid
Activity 1 Question 1 - pMB1 is a replication origin derived from an E. coli plasmid. pIM13 is an origin derived from a Firmicute plasmid. Why would the authors include this pair of origins of replication in the construct? (why 2 ORIs)
E.coli and Firmicutes use different machinery for replication and they both need to be able to replicate the plasmid
- Need E.coli ORI to keep the plasmif in E.coli
- Need Fimicute ORI to select for Firmucutes because it can only be used by Firmicutes and therefore the plasmid is only maintained by firmicutes
- Firmicute ORI also increases the liklihood that the plasmid is taken up by firmicultes
Activity 1 Question 2 - What is oriT? Why is it necessary for the proposed experiment?
OriT = needed for conjugation (OriT is recognized by the conjugation machinery)
Activity 1 Question 3 - The authors want to select for microbial recipients that have received the pMPM001 plasmid, but to also select against the E. coli donor so that it doesn’t overwhelm growth on the plate. What should be included in the media to select for pMPM001 recipients? How might the authors select against E. coli donor growth? Think about a general strategy, not a specific marker.
To Select fr PmpM001 (select for E.coli) = include the AB that the plasmid confers resistance to
To select for Firmicutes you can:
1. Put cells in growth conditions that favor firmicutes over E.coli
2. Exploit the differences in the biology of the donor E.coli vs. Firmicutes (ex. gram neg vs. gram pos)
3. Use Temperature senstive mutants for E.coli or E.coli that are auotrophic –> after select for E.coli with plasmid move the E.coli with the plasmid to a plate that lacks the molecule they can’t make or at the temperature that they can’t grow
4. Have two promoters in the plasmid (one for the E.coli and one for the Firmicutes to drive Antibiotic resistant genes)
- Under new condition the E.coli promoter is not active = E.coli lose resistnce = E.coli die and firmucutes with plasmid live
Activity 1 Question 4 - Through this experiment, the authors isolated roughly 60 bacterial strains from human stool. Given the known diversity of the gut microbiome, it seems clear that they did not isolate every organism, or even every Firmicute species. Brainstorm as many reasons as you can that an organism might not be isolated using the procedure outlined above (What series of events has to occur for a prospective recipient to grow as depicted? Each of these is a potential point of failure to isolate a given bug.). - OVERALL why might organsims not have been isolated
Many possibilities:
1. Some species fail to conjugate = they don’t ahve the plasmid = not resistnt
- Some cells did not get the plasmid
- Difference in the amount of speces = affects who gets conjugations
2. There is something in the body that the strains need to survive that is not in the media
- Example - Some bacteria might need to work with other bacteria (occurs in the body) and if they can’t have that interaction some of the bacteria might die
3. Species can lose the plasmid
4. Different strains use a different promoter OR translation is low in some strains (ribsomes not loading) so they don’t expresss the AB resistent genes
- Could be due to codon optimality (the AB resistent gene is not being tranlsated efficiently)
5. - Some bacteria might not co-exist in certain condition (might be difference in growth requirements)
6. Plasmid is mutated in a strain that has poor replication fidelity
7. Sample was not prepared well = lost some strains
8. Plasmid leads to a host defense response against foriegn DNA
- Cells degrade the plasmid OR the cells die when they have the plasmid
Transduction
Purpose - Allows for transfer for genes between bacteria (either from the chromosome or a plasmid) using bacteriophages
Although viruses do not frequently take up bacterial DNA the large number of viruses makes transduction successful at high frequencies
Overall Process - Uses bacteria phage to pick up pieces of bacterial donor DNA –> the phages will give the DNA to the recipient cell
Transduction Process
- Phage attaches to a donor bacteria and inserts genetic material into the cell
- Bacetria DNA is chopped into small peices –> Allows the virus to hijak the cell’s replication machinery
- Virus will replicate- When the virus replicates it will usuallly reincorprotae its won genetic information into its capsul BUT can also take up a chunk of bacteria DNA instead of the viral DNA
- Image – taking up bacteria DNA is show in cyan
- Phage can be incubated with the recipient bacterial strain –> allows for the transduction (transfer) of its passanger DNA into the recipient cell
- Passanger bacteria DNA = DNA that was taken up by the virus from the donor cell
- Since the virus lacks an intact virome once it is in the recipient cell – the bacterial genome is NOT cut up and INSTEAD the passanger bacterial DNA can be incorpoated into yeh recipient genome through homolgous recombination or can be retained as a plasmid
Fowards vs. Reverse genetics techniques in Bacteria
Reverse genetics – Mutagenisis + Alelle exchnage + conditional inactivation/overactivation
Foward Genetics – Spontenous/chemical mutagensis + transposons + Transposon mediated mutogensis + Tn-seq
Mutigensis
Directed mutogensis = makes chnages to gene’s sequence or structure
- Making intentional changes to the gene’s natural status
Things that you can do – Start with a gene that codes for a 3 domain protein:
1. Can delete the whole gene
2. Can delete domains or make truncations
3. Can get more specific and alter single amino acids (ex. Make point mutation)
- Do with Site-directed mutogensis + Error-Prone PCR
4. Can make fusions (Ex. Fuse gene to floruscent protein)
CRISPR in bacteria
CRISPR as a gene-editing technique is not widely available in most bacteria –> so allele exchange or other similar techniques are typically the most reliable way to make specific mutations
How do you asses the phenotype of a mutated gene
Best way to assess the phenotype associated with a mutant gene is to exchange the WT allele at its native locus for the mutant –> does using Allele exchange
Allele exchange
Overall - uses Homologous recombination to put information from the plasmid into the host chromosome
Purpose - engineer the chromosme of bacteria
- Example - make a gene deletion or a mutation or can integrate a locus with something that we want
Uses a suicide vector
Overall Process – Rely on host Homologous recombination to allow for the plasmid to recombine into the host chromosome THEN use a selection marker (that was on the plasmid) to select for cells that integrated the plasmid into the genome
What does the plasmid need to have for Allele exchange
- Mutant gene of interest
- Selectable marker (Ex. Kanomycin restsint KanR)
- Selects for bacteria that have plasmids that have integrated into the host chromosome
- Counter slectable marker (Example - SacB)
- For simple plasmid integration you don’t need SacB
- Non-function Ori
- Have a region that has homology to the region of the bacteria genome that you want to change
Image – blue gene of interst = region of homology between the vector and the bacterial chromosome
Why does the plasmid need to integrate into the genome in Allele exchnage
Things that make sure the plasmid integrates into the genome:
1. Non-fucntion Ori
- If it had an ORI then it would be able to be maintained extra chromosomally BUT by not having the ORI the only way to maintain the plasmid if by integarting into the genome
- Because the ORI is not function –> the plasmid will not replicate on its own –> therefore the plamsid is forced to integrate into the chromosmes in order to be maintained
- Selection for a selectable marker (Ex. Antibiotic resistnce)
- Kanomycin selection further forces plasmid integration
What do you need for plasmid to be intergted into the genome in allele exchnage
In order to integration to occur you need to include enough of the gene for the cell to “recognize” that the plasmid is homologous to the chromsome
The length of the gene could be enough BUT you OFTEN need to include regions upstream and downstream of the gene
- Regions upstream and downstream of the gene = homology arms
- Need homology arms if you are deleting the gene
Allele Exchnage - process
Overall - Entire allele exchange process entails 2 Homologous recombination events (Mutant allele will undergo homolgous recombination with the WT allele –> THEN the mutant allele will integrate into the genome)
Process:
1. Have 1 homologous recombination event between the mutant on the plasmid and WT allele on the chromosomes
- After 1 Homologous recombination even = have original WT gene and the mutated version that you introduced + ALSO have everything that was on the plasmid (Ex. Kanomcyin restence and SacB) in the host chromosome
2. Select for integrants by growing the cells on kanomycin (because have kanomyciin resistnt gene that was added to the chromses during HR event)
- IF you are preforming a simple plasmid integration THEN you do not need SacB and you stop here
- ALSO If you want to keep the things in the plasmid that is NOT the gene of interest in the chromosome THEN don’t need the counter selectable marker –> can just mainatin the plasmid in the chromosome using kanamycin resistence
3. Take intiation integrate and grow in absence of selectable marker THEN Have 2nd reocmbination event that allows the WT allele to be removed from the chromosome
4. Select for sucessful second crossover
5. Need to confirm that the mutant gene is present on teh chrosmoe using PCR or whole genome sequecesing or Wester blot
Allele Exchnage - Second recombination event
Have 2nd reocmbination event that allows the WT allele to be removed from the chromosome
- Needed IF you want to pop out the rest of the plasmid and ONLY keep the gene of interest/mutated version of the gene inserted in the host chromosome (done if you want to remove the Kan and SacB and WT)
2nd recombination event = the WT allele will cross over for a second time with the mutant gene (occurs on the other side of the mutation that it did the first time)
- NOW can’t select with Kanamycin because you lost the kanamycin resistance gene = use a counter selectable marker
- Technicaly recombination could occur on either side
Result of second crossing over event can yeild two options:
1. Mutant is left on the chromsomes and remove the plasmid (if reocmbination occurs on the other side of he mutation than it did the first time)
2. WT alelle is left on the chromosome (occurs If recombined on the same side as the first recombination event = get WT)
Image – shows the same sequence but rearnged in space
Allele Exchnage - Select for sucessful second crossover
Looking for cells whose chromosome lost the insertion casset using the counter selectalble marker
- Selecting for the second crossover
Process - Grow cells on sucrose and select against the procesnce of the SacB gene (selecting for thing that do NOT have SacB becuase SacB should be lost during second crossover)
- If have SacB then SacB makes sucorse toxic = cells that survive on sucrose do not have SacB/anything with SacB dies
- Anything that removed the rest of the plasmid = has no SacB = is sucrose resistnt
- Select against things that did not have second recombination event
END - cells should be Kan sensative and Sucrose resistent if they poped the plasmid out in 2nd recombination event
Allele Exchnage - Confirm the mutant gene is present
Need to confirm that the mutant gene is present on the chromosome using PCR or whole genome sequencing or Western blot
- Need to sequence the cells to verify that the WT alelle is no longer there (make sure you did not revert back to WT)
Why do you plate cells on sucrose after the second recombination event?
Answer - B – to isolate the cells that have lost the insertion cassette
Conditional gene inactivation/overactivation
Overall – changing gene product levels
Purpose - Used when you want to look at phenotypes that are associated with a partially knocked-down gene
- THIS is knockdown vs. allele exhcnage is KO
- Partial knockdown (inactivation) = partial or near total loss of gene expression
- Might do for essential gene OR to study the transition of high to low expression
Can ALSO over activate a gene –> express the gene at higher levels
- Ex. Enhance a particular phenotype
Example – want to do RNAseq at different points during depletion
Examples of conditional gene inactivation or over activation in bacteria
Inducible expression –> can be accomplished by placing a gene of interest behind a promoyer that is inducible by a small molecule
Example - Small molecule that can induce the promoter = IPTG ; Example gene = yfgA
- Just move the gene to downstream of the downstream gene and downstream of promoter that IPTG binds to
- Expression levels are affected by - Inducer concentration+ Expression from chromosome vs. integrating plasmid vs. replicating plasmid
Use of IPTG
Construct can be used to deplete an essential protein
- Need to deplete an essentilal protein because you can’t delete the gene
To deplete the essential protein –> grow the cells in the prescence of inducer and THEN wash out the inducer to stop transcription –> Protein will eventually be degraded = can observe the phenotype that results from loss of the protein
CRISPRi
Inhibits transcription
used for conditional gene inactivation
Altering gene function at the protein level
Used for conditional gene inactivation or over activation in bacteria
Example - Protein degradation can be induced in bacteria using the SsrA degradation tag system
Process of SsrA tag system - a gene of interest is tagged with ssrA –> THEN induce the expression of sspB –> ssPB will bind to ssRA tagged proteins –> Signals for degredation of the proteins
- SspB an adpater that binds ssRA tagged proteins
- Allows you to selectively control protein level on demand
Temperature-sensitive proteins
Temperature-sensitive proteins can also be used to selectively inactivate a protein at a particular temperature
Used to alter gene function at the protein level
Do bacteria have RNAi
There is no RNAi in bacteria — they do not have the machinery to generate siRNAs
Answer – E
Foward genetics in bacteria
- Sponetenous/Chemical mutogensis
- Transposons
- Transpson-mediated mutogensis
- Tn-seq (comparative transposon sequencing)
Sponetenous/Chemical mutogensis
Do sponetenous/Chemical mutogensis –> then hunt for mutants or supressor screen –> do whole genome sequencing after you get mutants to identofy the mutation
In bacteria we commonly perform spontaneous mutagenesis and less commonly use chemical mutagens to create a library of mutants
- Use spontaneous mutogensis for mutant hunt
WHY use sponetenous mutigensis:
- Mutation rate - ~10^-10 to 10^-9/nucleotide/generation; Genome size - 0.5 to 13 Mbp
- Ex. - Caulobacter crescentus – 4.2 Mb genome –> 3.5*10-10 mutations/nucleotide/generation –> ~1 in 700 cells acquires a mutation each generation (every ~90 minutes) –> get plenty of mutations without a mutagen
Transposon
Transposon = type of transerable DNA element that moves from a section of donor DNA to recipient DNA
Mechanism of transfer:
1. Transposase enzymes bind to the inverted repeats on the transposon
2. Transposase excse the transposon from the donor DNA using the invertes repeats
3. Transposase inserted the excised transposon into the recipient DNA
Transposon = usually contains an Antibiotic resistnace gene –> allows us to select for insertion in the new location
Transpson-mediated mutogensis
Start – need a phenotype of interest (Example – looking for mutations in genes that are repsonsible for growth in the prescence of an antibiotic )
Process:
1. Mutogenize cells with a transposon and transposase
- Insertion of the transpon into recipient DNA can lead to a delation and trancation mutations
2. Isolate the mutatesby plating on the novel antibiotic to select for mutants that can grow in the presence of the antibiotic + plate on KAN –> this selects for cells that have taken up the transposon
3. Pick individual cells and amplify across the transpson-chrosome junction
4. Preform sanger sequencing on the PCR products to identofy the mutant gene
How do you amplify across the transpson-chrosome junction
Overall - Use a primer for the transposon (foward primer) and library of random primers (reverse primer)
Because the transposon can be inserted anywhere in the genome we don’t know what sequence to use for the reverse primer = use a library of randomly created primers and one of these should be a good enough match to the inserted gene to yeild a PCR product
Generation of deletion/truncation vs. Generation of point mutations
To generation deletion or truncation - use Transposon mediated mutigensis
To generate point mutations or duplications = use spontneous mutogensis
Tn-seq
Comparative transposon sequencing
- Example - can compare WT vs. Mutant OR can compare insertions in different conditions
Purpose - screening technique that combines transposon-mediated mutagenesis with next-generation sequencing
- introduced transposon into WT strains vs. Mutant –> can see if there is a difference in the essential genes in the two conditions
Map all of the insertion sites accros the genome
Selecting for colonies in Tn-seq
Select for mutants that have a selectable marker with the transposon
- Each colony that grows will have transposon inserted somewhere in the genome
Can’t recover a colony IF the cell had a transposon in essential gene = IF the gene is essential there can’t be a transposon in it because then the colony would have been dead
Any genes that have transposons inserted in are non-essential
Image – Have inserstions in Gene C in WT (Gene C is not essential in WT) Vs. In mutant you have no insertions in gene C (In Mutant you can’t disrupt gene C )
- Shows synthetic lethal intercaton between the orginal mutation and the disrupting gene C
Tn-Seq Process vs. Transposon mediated mutogenes
Experiment the same as a simple transposon-mediated mutagenesis experiment BUT here we’ll pool all of the mutant clones instead of looking at them individually
Amplify using the same set of primers as before BUT we are amplifying all of the mutants at once to generate a massive pool of sequences where the transposon has inserted
Tn-seq process
Transposon-mediated mutagenesis experiment –> pool all of the mutant clones –> amplifying all of the mutants at once to generate a massive pool of sequences where the transposon has inserted –> Use next-generation sequencing to sequence this pool of PCR products simultaneously and map insertions to the genome –> THEN look at the frequency of transposon insertion at every locus in the genome to determine the essentiality of those genes
What does Tn-Seq show us
Tn-Seq = shows us how frequently a transposon inserts into every gene sin the genome –> we can use this to determine the essential genome of an organism
Genes that are essentilal - will have few or no insertions
- No insertions because the transposon would be knocking out essential gene function
Genes that are not essential = will have several insertions
- Have several insertions because knocking out these genes is permissible
Useful application of Tn-seq
Application - determine conditional essentiality
Purpose – finds genes that are required for growth under a particular set of conditions
Example - looking at genes that can grow in the presence of our new antibiotic
Process – compare transposon insertion frequencies under different conditions
- Compare insertion in no antibiotic or presence of antibiotic
Find - In one case - start with a gene with a high insertion frequency without antibiotic (it is non-essential when there is no AB) BUT THEN When antibiotic is added, we get no insertions indicating this gene has become essential under this particular condition (essnetial when there is AB)
Tn-Seq general idea
In genral in Tn-seq you are thinking about which genes become more or less important for fitness
Example - gene with many insertions now gets fewer insertions upon antibiotic treatment –> MEANS that when you have AB the gene becomes more important for fitness
Tn-seq use #2
Can use Tn-Seq in synthetic lethality screens - compare transposon insertion frequency in a wild-type and a particular genetic background
True
Methods of Antibiotic Resistence
- Bacteria Can encode a protein (enzyme) that can degrade the Antibiotic (produce a drug inactivating enzume)
- Mutation in the drug target in the bacteria so that the target can’t bind to the drug anymore
- Prevent intake of the Antibiotic by modifying the surface of the organism OR by activating eflux pumps that remove the Antibiotic from he cell
Activity 2 (JUST to set up for rest of flashcards)
You have a new antibiotic in hand with an unknown mechanism of action
- One E. coli strain (strain S) is susceptible to the antibiotic, whereas a second (strain R) is resistant.
You hypothesize that strain R is resistant, either because it has a mutation in the target or because it bears a drug-inactivating enzyme
Goals: identify the target in strain S and the mechanism of resistance in strain R
Assume: there is a single protein target that is essential for growth and present in both strains
How would you test that the mutation in pbp3 in strain R could confer resistance to strain S
Using Reverse genetics
Do allele exchange:
- Add Strain Sequence to Strain R –> See if strain R is now susceptible
- Add Strain R sequence to Strain S (replace copy on S with R copy) –> see if Strain S is now resistance
ALSO can add the non-conserved sequence by a plasmid (add a second copy of pbp3 gene fropm resistent strain) to the susceptible strain and see if that confers resistance on top of WT copy OR replace susepctive strain copy with resistent strain
- Plasmid would test for dominance
Expect that this would be dominant
Using foward genetics
Process - Grow cells –> allow for spontenous mutations to arise –> plate the cels on the Antibiotic –> Find strains that are NOW resistent –> Sequence the colonies with whole genome Sequences –> Can see what mutants induce Antibiotic resinet in those colonies
- The strains that are NOW resisntant might not have the same thing making them resistent as strain R
- Looking for point mutations
- Idetify the mutants by sleecting by growing them AB and sequence to know where the mutation was
DON’T want to use Tn-seq here because don’t want full loss of function because this is in an essential gene (want mutation that can decrease activity so that the drug can’t bind/target but can still work at a low level)
Use allele exchange to make sure that the mutation that we found was sufficient to confer resistence to S strain
NOW essential only in the presence of the drug NOW conditionally essentiallY)
Process - Do Tn-seq in R with or without the drug –> look for loss of reads in R with drug –> sequence for transposn insertion sites
- Loss of insertions in gene in R with the drug = indicates that those genes are essential in the presence of the drug (no reads in it = essential)
Looking for presence of reads in R without the drug and Loss of reads in R with the drug
- In Drug = have no insertion in the gene that confers resistance (in gene coding for the AB inactivating enzyme) ; without the drug have insertion in that gene
Mutations made = LOF because they are insertion
Would Tn-seq in R with or without the drug work on gene that is also essential in the absence of the drug
Would not work on a gene that is also essential in the absence of the drug because then BOTH drug and no drug would have no reads (couldn’t compare no inserstion and no inserstion if both drug and no drug had no insertions)
Last question - If you have multiple genes how would you show which confers resistnce
CRISPR 1 by 1 in the susceptible strain and see if that confers resistence OR can put each gene on plasmid and put the plasmid into the susceptible strain and see if it confers resistance
