Plasmids Flashcards
What is the origin of replication and what is importance of the role that the ori plays within the plasmid?
The ori is the sequence where replication is initiated.
It is important because it is the main determinant of the host cell range. Certain origins only work within certain species.
It also determines the copy number of the plasmid: this can either be high (e.g. pUC and ~500-700 c/c), medium (pET and ~40 copies/cell), low (e.g. pACYC and ~10 c/c) or ultra-low (e.g. RP4 and ~1-2)
The ori also determines whether two plasmids are compatible. Two plasmids would have to have different origins for them both to be maintained in a cell.
Ori can also be conditional - it may be temperature sensitive, host specific etc. This is useful in genetic manipulation as you can control replication using growth conditions.
Describe the process of origin incompatibility and why two plasmids with the same ori cannot be maintained.
When two plasmids have the same (or closely related) origin of replication, the replication machinery could replicate one plasmid more over the other, simply by chance. The copy number cap will apply to both of them regardless of how many of each are present. Therefore, when a cell divides, there will be an unbalanced split of the two plasmids. Through continual cell division, this process will exponentially increase the number of that plasmid because there are more of them and so they are more likely to be replicated. The other plasmid will eventually be lost over time from this colony.
If the copy number is high, it will take longer for the loss of one of the plasmid because the chances of one being replicated more than the other is then lower.
Describe the three components of plasmid partitioning and the process by which it occurs.
There are three components involved in plasmid partitioning:
- parS is a DNA sequence carried by the plasmid
- ParA is an ATPase protein. When bound to ATP, it can bind to DNA non-specifically.
- ParB is a protein that binds to DNA specifically at parS.
During cell division, ParA-ATP binds to the chromosomal DNA non-specifically, along the whole chromosome from one pole to the other. ParB, which binds to parS on the plasmid, finds a ParA-ATP and binds to it. When this happens, it triggers ParA to convert ATP to ADP. ParA-ADP then dissociates from both the DNA and ParB. This leads to ParB searching for more ParA-ATP to bind to- which will mean the few ParB-parS on the genome start to move toward the poles.
Plasmids present a metabolic burden for bacteria to maintain and upkeep. Therefore, they usually provide a beneficial function for the bacteria. Describe some of these benefits and an example of each.
Antibiotic resistance: this enables bacteria to persist even when in a toxic environments. E.g. an RK2 plasmid found in bacteria from an outbreak of antibiotic resistance in 1969 found that it contained ampicillin, tetracyclin, kanamycin resistance. These have likely arisen from transposons - movable DNA elements - that have moved between bacterial cells.
Virulence genes: This allows bacteria to become pathogenic and grow in new environments where there are no competitors e.g. within plants or animal hosts. E.g. shigella flexneri contains a virulence plasmid which encodes the type III Secrety System necessary for infection.
Metabolism genes: allows bacteria to utilise addition types of nutrients. E.g. plasmids containing chlorobenzoate catabolism genes allow bacteria to metabolism PCBs, this very toxic pollutant that used to be used in coolants.
Antibacterial competition systems: enables bacteria to kill competing bacteria e.g. E. coli can carry ColE1 that contains the gene for colicins.
What are addiction modules in plasmids and why are they termed as a ‘selfish’ gene? Give an example.
Addiction modules are toxin/anti-toxin systems that ensure a bacteria keeps a plasmid. It contains two genes, often overlapping, that encode for a stable toxin and an unstable anti-toxin. Because the anti-toxin is unstable and prone to degradation, if the plasmid is lost from the bacterium, the toxin remains in the cell and kills it.
An example of an addition module system is the hok/sok system from plasmid R1. In this case, hok and sok genes are one gene that is transcribed both ways. If the plasmid remains in the cell, sok mRNA neutralises hok mRNA by hybridising to it (as they are complementary sequences) and are both degraded. But if the plasmid is lost, the toxic hok mRNA does not hybridise with sok, and is translated into a hok protein, causing cell death.
What are some of the key features present on a plasmid vector used for recombinant DNA technology?
Essential -
- Origin: start of replication. Have to make sure it is compatible with the host and possibly other plasmids.
- Selection marker: usually containing antibiotic resistance and allows for colony screening to identify colonies that have been transformed correctly.
- Multiple cloning site MCS: different restriction sites to allow for insertion of DNA fragments.
Optional -
- Promoter/terminator: necessary for transcription if you want to produce a protein.
- origin of transfer: allows mobilising of plasmid from one cell to the other e.g. during bacterial conjugation.
Counter-selection marker: negative selection or conditional selection of the plasmid.
What are three examples of colony screening that can be used to ensure bacteria have been transformed with the plasmid vector?
Blue-white screening: A gene for an enzyme can be used, such as beta-galactosidase in pUC18. This enzyme cleaves a X-Gal protein and forms a blue pigment. The MCS is embedded inside the gene for the enzyme so if the DNA fragment is correctly inserted, beta-galactosidase will no longer be produced and the colonies will turn white. This is otherwise known as alpha-complementation.
Fluorescence screening: same as blue-white screening but with GFP instead.
Toxic gene product: A gene that produces a toxin that will kill the cell if the insert is not put into the plasmid. Therefore, only colonies with the gene insert grow.
What are shuttle vector? Describe its function and features. Give an example.
A shuttle vector is constructed so that it can propagate in two different host species. This is especially used between eukaryotic and prokaryotic in order to be able to manipulate the DNA in E. coli so then it can be transfer into a more difficult or slower to use organism e.g. yeast.
For example, YEp351 is a shuttle vector used for E. coli and yeast. It contains:
- OR of replication for both species.
- Selection markers for both species.
- MSC
- Bacterial promoter.
What are suicide vectors? Describe its function and features.
Suicide vectors work under the principle that they enter host cells where they cannot be replicated due to incompatible replication origins, but there is selective pressure (e.g. antibiotic environment) that forces it to undergo a single homologous recombination event between two homologous pairs, that recombines the whole plasmid into the host cell genome. This allows the cell to then use the antibiotic resistance marker on the vector. This can also work by using conditional origins, which don’t work under certain conditions e.g. temperature sensitive.
This can be used for allelic exchange, whereby the plasmid contains a mutant version of the wild-type gene. Recombination is allowed to take place between the plasmid and host cell genome, and so the mutation is introduced. The cells are then screened for ones with the mutated gene and experimented on.
It can also be used for transposon mutagenesis. This is where the transposon is designed to insert into a part of the chromosomal DNA that will disrupt a gene. The resulting cells can then be studies to observe the mutants and the varying phenotypes presented when that gene is interrupted.
https://bitesizebio.com/41461/old-reliable-two-step-allelic-exchange/#:~:text=The%20idea%20behind%20suicide%20vectors,understandably%2C%20can’t%20replicate.
