Plasmids Flashcards
What are 3 things plasmids are vs chromosomes?
What is the ori?
What else does it determine?
What is required for replication initiation?
Smaller, not essential for normal growth, mobile
Dictates copy number - how many copies plasmid present in a cell
Plasmid compatibility - machinery, resources, proteins etc
Exceed threshold amount of initiator for binding & de-annealing DNA in AT rich area
What does copy number depend on? 3 things
What are the uses for a high copy number?
What is the problem?
What are the uses for a low copy number?
How can you influence copy number?
How can a plasmid achieve low copy number unintentionally?
What else does copy number depend on?
- ori (& proteins required)
- size of plasmid & insert - larger = lower number as high metabolic burden
- culture conditions - metabolic burden
High protein yield
Protein aggregation & deficient post-trans modifications = metabolic burden
Express a toxic product & mutant studies - to minimise effects
Temperature shift (bacterial growth rate) & chloramphenicol (antibiotic inhibiting bacterial protein synthesis & DNA replication/division)
Large DNA insert is a natural metabolic burden - cells become less efficient & growth slows
Initiator proteins (repressor proteins which impact initiator directly)
What is the problem with plasmids having the same ori?
What happens in DNA replication & binary fission?
Compete for same resources, initiator proteins & machinery
Cells can’t differentiate between two different plasmids - so mixture of both plasmids or only 1 can end up in daughters
What two mechanisms could you use to maintain a plasmid in a cell?
What are the two mechanisms for even plasmid partitioning?
In what type of plasmids are these mechanisms encoded?
- Make it difficult to lose the plasmid (addiction module)
- Make it a selective advantage to keep the plasmid
Filament formation
Brownian ratchet
FF = R1
BR = pB171
What do the following do in filament formation:
ParM protein
ParR protein
ParC gene
What are the 4 steps for the filament formation mechanism in the Type II ParMRC system?
Filament forming ATPase motor - monomers that form filaments with ATP & nucleation
DNA-binding adaptor protein - binds to ParM to form filament
Partition site that ParR binds to
- ParR binds to parC on plasmid sequence
- ParM binds to ParR in it’s filament state and ParM will also bind to the other ParR-parC complex on the other plasmid
- Using ATP, the ParM filament will extend & segregate plasmids by bidirectional elongation
- Push 2 copies of plasmids to opposite poles of cell - both daughters contain 1 copy
What do the following do in the Brownian Ratchet mechanism:
ParA
ParB
ParC gene
In what motion do the plasmids partition?
What are the 5 steps?
ATPase - binds DNA when ATP bound (ParA-ATP)
Binds to parC & ParA-ATP to activate ParA atpase activity
sequence on plasmid
Wrench
- ParA-ATP binds to the chromosomal DNA
- ParB binds to the plasmid via the parC sequence & binds to ParA-ATP (on the chromosomal DNA)
- This activates ParA to convert the ATP into ADP
- ParA-ADP releases from the chromosomal DNA & ParB (on the plasmid)
- The ParB still on the plasmid continues to look for more ParA-ATP on the chromosomal DNA to bind - so that the plasmid and chromosome move together away from the other pair
What is counter selection?
How can antibiotic resistance be used?
Metabolism?
Virulence?
Bacteriocins/cell-cell antagonism?
Addiction modules?
What are some of the negatives of using virulence & bacteriocins?
How does the hok/sok toxin/anti toxin system work?
Using selective advantages to isolate cells with DNA of want
Survive on antibiotics that other cells naturally cannot
Contain genes to breakdown usually toxic chemicals in environment to survive
Encodes virulent genes with toxins, secretory machinery to survive in other hosts
Produce toxins/bacteriocins that kill off competitiors (e.g colicins & E. coli)
Toxin/anti-toxin system where anti-toxin mRNA required to degrade toxic mRNA
Virulence - very large plasmids (burden)
Bacteriocins - could target own cell
Stable hok (toxin) mRNA & unstable sok mRNA form duplex for degradation when both genes expressed. If plasmid is lost, sok mRNA degraded so hok mRNA translated into toxin
What is the requirement for a plasmid in molecular biology?
What are the other options?
What are the + and - of blunt cloning?
What is TA cloning?
What are the -?
origin of replication
MCS, selection marker, promotor/terminator, origin of transfer (transfer from 1 cell to another), counter-selection marker
Easy
No specificity with DNA & plasmid
Using Taq polymerase to add A at 3’ end to PCR product - requires T overhang on vector backbone
No orientation/symmetry
What are the + and - of restriction endonuclease cloning?
What is golden gate cloning?
What are the + and -?
What is seamless cloning?
What are the + and -?
+ unique overhangs & limits number of orientations
- requires designed restriction sites
Using engineered restriction sites outside of the recognition sequence to have any overhang sequence
+ allows cloning in correct orientation due to high specificity
- need recognition sequence to generate overhangs
Overlapping PCR sequences to assemble into vector
+ correct orientation
- overhangs need to be long
How does the white/blue screening mechanism in pUC18 vectors work?
What is a toxin alternative to screening?
How is the pET expression vector controlled?
pBAD expression vector?
What is a leaky promotor?
Beta galactosidase gene in MCS - if present (unsuccessful cloning) degrades X-gal to produce blue colonies, if cloned successfully gene is disrupted & colonies remain white
CcdB gene in MCS - when DNA inserted toxin no longer expressed
Lac system - add inducer lactose to over-express (induce the operon)
Arabinose which binds to AraC transcription factor to allow RNAP binding - if arabinose not present, AraC binds to aral1 & araO2 to form loop in DNA to prevent RNAP binding to promotor
One that doesn’t totally control/regulate gene expression (e.g attentuation of trp operon prevents leak)
What is site-specific recombination?
What is a suicide vector?
What are its applications?
What does it require to show the plasmid is not retained?
How can genes be stably expressed?
DNA strand exchange between sites of high homology by recombinase
Vector used to enter a cell but not stay
Gene replacement/allele exchange, transposon mutagenesis & transient expression of genes
Counter-selection marker
Integration into host chromosome by changing conditions such that they can no longer replicate as its own entity
What is homologous recombination?
What is the difference between homologous recombination & site-specific recombination?
What happens with a longer stretch of homologous sequences in recombination?
How can suicide vectors utilise either of these?
How do vectors undergo homologous recombination with the host chromosome?
Chromatids with similar sequences recognised by RecA & swap specific sequences
Homologous = molecules of similar sequences (like DNA & RNA) & recognition by RecA
Site = specific sequences in DNA (recombinase)
Higher frequency recombination occurring
Integrate into host chromosome
Homologous sequences recognised by RecA - allowing other factors to assemble for recombination
What is transposon mutagenesis?
What does it require?
What kind of genes do the transposons usually carry?
Using suicide vectors & transposons to bring translocate genes into the host chromosome
Suicide vector with transposon, transposase & antibiotic selection for both the backbone & transposon
Antibiotic resistant genes
Give the 3 types of horizontal gene transfer & a brief description.
What does conjugation require?
What is the problem with conjugative systems?
What other specialised systems help with conjugation?
- Transformation (free DNA in environment taken up)
- Transduction (DNA in phages & infection)
- Conjugation (mating pair formation & DNA transfer)
Direct cell-cell contact with donor & recipient
Have different host ranges - F is very narrow (e. coli) and RP4 (gram -ve) but Ti used in eukaryotic plants
Type 4 secretion system
How was conjugation first found?
How is the F pili formed in E. coli? 4 steps
How does the F pili work?
How is RP4 pili different to F?
How does a mating pair form with F pili?
RP4 mating pair?
2 Tubes with bacteria & different nutrients in each. 3rd tube introduced with mixing of nutrients & bacteria - led to colonies while individual nutrient tubes didn’t (sharing of plasmids led to survival)
- Pro-pilin polypeptide made in protein synthesis & inserted into membrane
- processed & trimmed to form mature pilin
- pilin attaches to a phospholipid
- pilin-phospholipid is assembled into pillus of T4SS
Extends out the donor cell via the T4SS, attaches to recipient & retracts to pull cells into contact
- Shorter (doesn’t extend as far)
- Higher efficiency but doesn’t capture bacteria same way
- in aq solutions, efficiency is lower
- rigid pilus - broken easily
Forms translocation channel with hydrophilic centre allowing solubles & DNA through
Much closer cell-cell contact - tight interactions
What is a relaxosome?
What is it made up of?
What are the 3 steps of its formation?
How is it bridged to the T4SS?
The relaxase, helicase & transesterase domain are involved in the relaxosome. What do each of them do?
What is the site that DNA transfer begins on transferred molecule?
Protein complex that facilitates conjugation - allows for DNA transfer & complimentary strand synthesis
Relaxase (binds to oriT), oriT (where transfer begins), accessory proteins
- Relaxase binds to the oriT (origin of transfer)
- Nicks the DNA (cuts one strand) and unwinds DNA at this sequence (oriT)
- Accessory proteins bind to stabilize this complex/bend the DNA
Coupling protein
Relaxase = cuts nic site for complimentary strand synthesis
Helicase = unwinds & separates DNA after initiation
Transesterase = forms covalent bond with 5’ end of nick
nic site (encoded within oriT)
Why is T4SS believed to act like a switch?
In 3 steps, how does the DNA get transferred into the recipient?
In what form is the DNA being transferred?
What do SSP proteins do in this case?
Goes from pilus biogenesis to DNA transfer mode
- Coupling protein inserts into T4SS & displaces BII domain
- Displacement allows relaxosome to bind to plasmid for transfer
- Relaxase transferred through into pilus first - carrying bound DNA in a string-like movement
Single stranded (helicase domain)
Bind to single-stranded plasmid DNA to prevent its degradation
How does the recipient cell synthesise the complimentary strand? 2 steps
How does the donor cell synthesise the complimentary strand?
What else can be transferred in conjugation/DNA transfer?
What is their problem/solution?
What is the final step of conjugation?
- Relaxosome on 5’ end joins up with the 3’ end to make single stranded circular DNA
- DNA replication of lagging strand occurs - primers, Okazaki fragments, ligation
Uses the 3’ end to serve as the primer for DNA replication & lagging strand synthesis
Integrative conjugation elements
Don’t have ori - so integrate into chromosome & form circular DNA (not plasmid)
Donor & recipient separate
Hfr (high frequency of recombination) bacteria have a different response to conjugation. What happens before DNA transfer with a plasmid in the cells?
What happens after DNA conjugation has stopped in recipient?
How can you use Hfr (strep susceptible) and F-(strep resistant) cells to show chromosome mapping with nutrient gene A+?
Due to hfr, F plasmid integrated into host chromosome
Transferred conjugated DNA either is degraded or undergoes homologous recombination
If conjugation occurs between Hfr carrying A+ and F-, can be plated on selective media & should survive. Donor Hfr cells die on strep medium
What is fertility inhibition?
What happens if both F and RP4 plasmids are present in a cell?
Why?
What is an exclusion factor?
What are the 2 types of exclusion? Give brief descriptions
Which of these mechanisms do F or RP4 plasmids have?
Type of plasmid incompatibility where donor bacteria can only transfer a few conjugative plasmids - competition
Plasmid F is transcribed/conjugated
Conjugative elements encode machinery to interfere with transcription & DNA conjugation of other plasmids
Prevent recipients being donors of the same conjugative plasmid - wasteful
Surface: prevents mate pair formation/cell-cell contact
Entry: prevents DNA transfer completing
F have both, RP4 has entry
What are the +ve and -ve of transformation?
Conjugation?
Transduction?
+ don’t need specific machinery like T4SS
- need competent hosts & low frequency event/recombination
+ scales up low frequency events, DNA transferred linear so higher efficiency
- doesn’t work as less specific, need linear DNA for recombination
+ good to transfer 1 chromosomal mutation
- difficult to use for multiple processes
What is a shuttle vector?
What does it require?
What is triparental mating with RP4 helper?
When can this be used?
What is the problem with this?
How could you solve this?
What is a specialised donor strain?
Vector that allows transfer of cloned DNA between different organisms (e.g e. coli to higher eukaryote)
1+ ori, selection marker for both hosts
RP4 plasmid (helper which codes for conjugation) inserts into donor to make cloning cell mobile to transfer into recipient
If cloning vector in donor needs to be conjugated into recipient & overcome plasmid mobilisation e.g if members of same incompatibility group
RP4 plasmid could transfer into recipient instead of the cloning vector
Make RP4 temperature sensitive such that its initiator proteins are only active under certain temperature - inducing temperature conditions into recipient with both RP4 & cloning vector will prevent RP4 working in them
Donor strain with RP4 integrated into chromosome so cloning vector can transfer into recipient without RP4 transferred
What is two-step allelic recombination?
What happens after the first recombination?
What happens after the second recombination?
What are the two outcomes after the second recombination event?
Introducing a shuttle vector with mutant gene to insert into host chromosome
Grow bacteria on antibiotics to ensure that organisms that survive have the antibiotic resistant gene from homologous recombination
Grow bacteria on sucrose (counter selection) as sacB in vector backbone causes cells to burst - so kills off cells with vector remaining
Cells with either the original wild-type chromosome, or the mutant chromosome
What are the requirements of the suicide vector for transposon mutagenesis?
What is required of the donor cells?
What happens following conjugation?
How can you select for the cells with the transposon integrated into their chromosome?
Why will the vector not be retained?
How can you test that the suicide vector hasn’t been retained?
Why would you not want to retain the vector?
How can you test to confirm it’s transposition and not a recombinant event?
- transposase
- transposon
- antibiotic selection on plasmid backbone
- antibiotic selection for transposon
Contain transposon suicide vector with the inducer to induce transposase promoter
Suicide vector inserts into recipient cells & transposase is expressed - facilitating transposon to jump to new location
Grow cells on antibiotic selection for transposon (will be integrated in the chromosome)
No longer has the inducer so cannot replicate - inducer only present in donor
Counter-selection antibiotic resistant marker
Contains transposase genes - carry on putting transposon into different locations
- see if still sensitive to antibiotic (transposon resistance marker)
- counter selection marker on vector backbone
What is site-specific integration?
What else is required apart from the CTX plasmid coding for the phage site?
What does it produce?
What feature about it allows for its transient expression for only when needed for CTX?
Using phage integration sites present on both plasmid & chromosome to integrate genes
Transient expression induction (FLP)
Flp recombinase to remove plasmid backbone so only have gene of choice in chromosome
Temperature sensitive - growing cells at a temperature to transiently produce FLP to remove backbone in chromosome - but at normal cell temperature 37C FLP is not expressed
Agrobacterium are a pathogen to plants. What are the two plasmids in the transfer DNA binary system?
What is a Ti plasmid?
Why is the transfer DNA binary system more favoured over using a Ti plasmid alone?
What does the Ti plasmid naturally do in bacterium?
Helper plasmid: virulence mechanism from Ti plasmid with conjugation system to transfer T-DNA into host plant
Shuttle/binary vector: carries T-DNA genes, antibiotic marker (backbone), counter-selection (plant)
From agrobacterium - transfers T-DNA from bacteria using T4SS (virulence) into eukaryotic plants
Ti plasmids are very large & metabolic burden
Induce tumour formation
What do initiator proteins do?
What else is copy number dependent on?
Bind at a site near the ori - allows for contortion DNA/unwinding to allow DNA polymerase for replication to bind
levels initiator protein present & repressors of initiators
