3 - Plasmids

What is the importance of plasmids?

1. Can use as cloning vectors to introduce foreign genes into bacteria. 2. Method for horizontal gene transfer of genes, including AB-resistance in bacteria.

Describe the ParA partitioning system and provide an example.

- ParA-ATP dimers bind to CHROMOSOMAL DNA at random positions - leads to ParA filament formation. - ParB dimers will bind to parS site on the PLASMID. - When ParA is associated with ATP, the ParB-parS (and accompanying plasmid) can associate with an end of the ParA filament attached to chromosome. - This interaction signals ParA dimers to hydrolyze ATP to ADP, which releases ParA from the DNA. - As the first ParA dimer is released, the ParB-parS complex binds to the next ParA dimer in the filament. "Hops." - When the end of the filament is reached, the plasmid dissociates from it and can move to a new filament in a different location. The ParA dimers are also released and can move to a new location, but the exchange of ADP to ATP is a slow process that favors ParA diffusion to a new location. - Such a system will naturally distribute the plasmids away from one another in the cell. Examples of a plasmid that use this method of partitioning is the F plasmid.

Described the ParM partitioning system and provide an example.

- Plasmid contains two protein coding genes: ParM and ParR. - Also contains a parC site that can bind to ParR. - Actin-like ParM-ATP protein forms a filament (via polymerization) that pushes the plasmids to the cell poles. "Pushes" ParR at the the cap-like ParR-parC complex. - When ParM-ATP is hydrolyzed (ATP becomes ADP), depolymerization occurs, leaving plasmids at the poles. An example of a plasmid that uses this method is the R1 plasmid.

Describe copy number regulation using RNA1 and provide an example.

- RNA II is required as a primer for plasmid DNA replication. -The small plasmid-encoded RNAI is partially complementary to RNA II, and will bind to it if present. This initial pairing is weak, known as the "kissing complex." - ROP's presence ensures the binding of RNA I and II is strong ("hug") and so RNA II cannot bind to the plasmid DNA that is to be replicated. Since RNAI is synthesized from the plasmid, more of is made if [plasmid] is high. Same for ROP. Example: ColE1 plasmid (16 copies).

Describe copy number regulation via coupling and provide a few examples.

- RepA genes (for replication initiation) are found at the ori region. - The ori region contains 3 repeated sequences (iterons - R1, R2, R3) that can be bound by RepA. - If there are too many plasmids, there is a higher concentration of RepA in the cell, and they can form dimers with each other (while attached to the plasmids at the iteron sequences).

Describe rolling circle replication and provide examples.

- RepA protein binds to the double-stranded origin (DSO) of the plasmid DNA. Contains a palindromic sequence that bps, and RepA nicks one of the strands. - RepA holds on to the 5'-end of the nicked strand (via a tyrosine). - DNA polymerase III uses the free 3'-end as a primer and replicates a new strand around the circle, pushing the old strand out of way (still attached to RepA, and helicase unwinding it). - Once finished, the 5'-end attached to RepA is returned to it's 3'-end (now displaced), sealed with ligase. Have one ds-stranded, circular DNA and one ss-stranded circular DNA. - the ss-stranded DNA is replicated by DNA pol III and we end with two identical ds-stranded circular plasmid DNAs. https://www.youtube.com/watch?v=FhcZLqvs5yg

pUC plasmid.

- modified to have high copy numbers (no ROP and a more efficient RNAII (via a point mutation). -selectable Amp-r gene. ColE1 origin or replication.

What are some mechanisms of plasmid copy number regulation?

- regulation via RNAI - regulation via coupling.

What are the two types of plasmid replication?

- theta replication. - rolling circle replication.

pVLT31

-Broad range ori region. - May also contain a mob site, allowing the plasmid to be mobilized to other bacteria.

Describe incompatibility.

-Plasmids that cannot coexist stably in the same host together are said to belong to the SAME incompatibility group. i.e., they are incompatible. Eventually, after several divisions, one plasmid will be lost. - If two plasmids can be stably maintained in the same host they are from DIFFERENT INCOMPATIBILITY GROUPS. - If two plasmids share the same partitioning system, they will be incompatible. - If two plasmids share the same mechanism of replication control, they will be incompatible. *** Incompatible because they are similar.

pBAC

Bacterial artificial chromosome. - only 1 copy - Large (300 kbp). - MCS contains HindIII and BamHI restriction sites. - F plasmid ori (oriV).

What are endonucleases?

Bind to ds-DNA and cut at specific sites.

pWWO

Catabolic (breakdown) plasmid from Pseudomonas putida that encodes genes for utilization of aromatic hydrocarbons such as toluene as a carbon and energy source.

What are shuttle plasmids ? Please give an example.

Generally have 2 origins of replication, and so they can be replicated in more than one organism. - Note that they must also contain selectable genes that can be expressed in each organism. Example: YEp13: - contains ori and Amp-r (can replicate and be selected for in E. coli. Also contains 2um origin and LEU2 (can be replicated and selected for in yeast).

Plasmids vs. genomic DNA.

Genes encoded within plasmids are usually not essential for the growth of the bacteria under normal circumstances. Can confer advantage to bacteria host only under certain conditions.

The plasmid replicates BLANK of the chromosome.

Independently.

What is MCS?

Multiple cloning site. Contains restriction enzyme recognition sites.

Plasmid conformation, size and placement on agarose gel.

Nicked, circular plasmid: Largest, will be the top band. Linear plasmid: will travel vertically, in the middle of gel. Supercoiled, covalently closed circular DNA: smallest, will be found on the bottom of the gel.

What is partitioning and what are the two methods discussed in class?

Partitioning is when the plasmids are translocated to poles of the bacteria to ensure that daughter cells each receive a copy of the replicated plasmid. - ParM system. -ParA system.

Explain a method of plasmid maintenance.

The plasmid (such as the F plasmid of E. coli) can use the tox-antitox system. - 2 components present in plasmid (can be RNA or proteins). - one functions as a toxin, and the other as an antitoxin/antidote. -Both are made in the cell, with the antitoxin inactivating the toxin. -If the plasmid is lost during division, neither components will continue to be made. - The remaining toxin (present in cytoplasm from mother cell) is more stable than the antitoxin, so eventually the antitoxin is degraded and the toxin is left to kill the cell. - Ensures the survival of the plasmid.

Describe insertional inactivation.

The plasmid contains the lacZ gene, producing the enzyme beta-galactosidase that can cleave X-gal and form a blue color. - DNA inserted within lacZ gene will produce white colonies. - Also inducible - add IPTG or lactose.

Describe theta replication and provide examples.

Very similar to chromosome replication - the opening of the two strands of DNA at oriV.RNA primer begins replication either uni- or bidirectionally. Examples: ColEI and F plasmids in their vegetative cells (i.e., not conjugation).

A mob site allows...

allows the plasmids to be immobilized into other bacteria.

Host range and copy number are usually determined by...

ori region of the plasmid. (oriV).