Plasmid Types: F and R Plasmids in E. coli

Plasmid Types

• Plasmids are classified based on their main functions.

F-plasmids (Fertility Factors)

• F-plasmids, also known as F-factors, are well-characterized plasmids that play a crucial role in bacterial conjugation in E. coli.
• F-plasmids are circular dsDNA molecules, 99,159 base pairs in length.
• One region of the F-plasmid regulates DNA replication via rep genes.
• A second region contains transposable elements like IS3, Tn 1000, IS3, and IS2 genes, which allow it to function as an episome.
• A third region, called the tra region, contains tra genes and promotes plasmid transfer during conjugation.
• E. coli F-plasmids are an example of F-plasmids.

R-plasmids (Resistance Plasmids)

• R-plasmids are the most common and well-researched plasmids, which confer resistance against antibiotics and growth inhibitors.
• R-plasmids contain genes that encode enzymes that can destroy or modify antibiotics.
• Plasmid R100, a 94.3 kilobase-pair plasmid, carries resistance genes for sulfonamides, streptomycin, spectinomycin, chloramphenicol, tetracycline, and mercury.

Virulence Plasmids

• Virulence plasmids increase the pathogenicity of the host bacterium.
• They improve the bacterium's ability to resist host defenses or produce toxins.
• Ti-plasmids in Agrobacterium tumefaciens induce crown gall disease in plants.

Col-plasmids

• Col-plasmids enable host bacteria to kill other bacteria by secreting bacteriocins.
• Bacteriocins kill cells by creating channels in the plasma membrane, increasing its permeability and also degrade DNA/RNA or attack peptidoglycan to weaken the cell wall.
• Bacteriocins are effective only against closely related strains.
• The Col E1 plasmid of E. coli codes for colicins, which kill susceptible E. coli strains.
• Some E. coli Col plasmids code for cloacins, which kill Enterobacter species.

Metabolic Plasmids

• Metabolic plasmids, also known as degradative plasmids, have genes encoding enzymes that break down unusual substances.
• Substances degraded include toluene, pesticides (like 2,4-dichloro-phenoxyacetic acid), and sugars like lactose.
• The TOL (pWWO) plasmid of Pseudomonas putida is an example.

Plasmid Nomenclature

• The "p" stands for plasmid.
• "BR" indicates the laboratory where the vector was constructed.
• In the case of pBR322, BR refers to Bolivar and Rodriguez, the scientists who constructed the plasmid.
• The number "322" distinguishes this vector from other vectors developed in the same laboratory at the time of construction in 1977.

Plasmid Properties

• Plasmids are defined as extra-chromosomal genetic elements, mainly found in bacteria but rarely in eukaryotic organisms.
• Bacterial plasmids are circular, double-stranded DNA molecules that contain genes controlling diverse functions.
• The sex-factor or F plasmid of E. coli K12 was the first plasmid discovered and can exist in two alternative states: it can remain free in the cell or integrate into the E. coli chromosome, such plasmids are known as episomes.
• Plasmids replicate independently and can code for their own transfer, and pick up/transfer bacterial chromosome genes.
• Plasmids can carry genes or gene sequences coding for antibiotic resistance, which confers a selective advantage to their host in a competitive environment.
• Plasmids vary in copy number, which refers to the number of copies of a specific plasmid within a cell.
  • Smaller plasmids generally have a high copy number, while larger plasmids have a low copy number.

Plasmid Incompatibility

• Plasmid incompatibility is the inability of two plasmids to coexist stably in the same bacterial cell line over generations which is more common for closely related plasmids.
• Incompatibility often occurs when both plasmids possess a replicon with the same Rep protein specificity or controlling elements.
• Unrelated plasmids, such as F plasmids and ColE1, can coexist because they belong to different incompatibility groups.
• Plasmids from one incompatibility group exclude each other in replication but generally coexist with plasmids from other groups.

Plasmid Copy Number

• Plasmid copy number indicates the average or expected number of copies per host cell and can be low, medium, or high.
• Copy number varies depending on:
  • The origin of replication (ori) and its constituents (e.g., ColE1 RNA I and RNA II).
  • The size of the plasmid and its insert as larger plasmids/inserts need more metabolic resources.
  • Growth conditions such as aeration, temperature, culture volume, antibiotics, and medium can all affect copy number.
  • The freshness of the culture where freshly streaked bacteria have higher copy numbers if picked from a single colony without direct subculture.
• Also freshly transformed cells yield higher plasmid levels than colonies streaked from glycerol stock.
• Incubation for 12-16 hours also increases copy numbers as bacteria reach stationary phase.

Cloning Vector pBR322

• "p" denotes plasmid, "BR" is for Bolivar and Rodriguez, and "322" is a numerical designation.
• pBR322 was constructed in 1977 in Herbert Boyer's lab at the University of California, San Francisco.
• Its origin of replication is pMB1.
• pBR322 is a synthetic plasmid that was the first artificial plasmid to be constructed.
• pBR322 is composed of 4362 bp DNA from col El.
• It can be amplified to 1000-3000 copies per cell.
• It contains an origin of replication (ori), two selectable marker genes (ampicillin (ampr) and tetracycline (tetr) resistance), and unique restriction sites for 20 restriction endonucleases.
• 40 unique restriction enzyme genes are located within the TetR gene, and 6 restriction sites are within the AmpR gene.
• Insertion of a foreign DNA segment into the tetracycline resistance gene will inactivate it, causing the recombinant plasmid to only protect cells against ampicillin.
• Colonies appearing on ampicillin-containing medium are transformed colonies with the newly inserted DNA molecule.

Advantages of pBR322

• pBR322 is widely used due to its manageable size.
• Antibiotic resistance genes simplify the selection process of recombinants.
• Multiple restriction enzyme sites enhance the plasmid's compatibility.
• A high copy number is advantageous in genetic engineering.

Disadvantages of pBR322

• pBR322 has high unwanted mobility through the F pilus.
• Inserts are limited in size.
• Screening can be time-consuming.

Phage Lambda (λ) as a Vector

• Bacteriophage λ infects E. coli cells and the DNA is 48.5 kb long.
• The ends of λ phage DNA have cos (cohesive) sites that consist of 12 bp cohesive ends.
• The cos ends allow for circularization within the host cell.
• Large DNA fragments (up to ~20 kb) have their nonessential lambda DNA removed.
• Recombinant DNA is then packaged within viral particles in vitro, which are then allowed to infect bacterial cells (E. coli) plated on agar.
• The recombinant viral DNA is replicated within the bacteria, and genes continue the lytic cycle of cell lysis.
• Phages of lysed bacterial cells creates plaques on a bacterial lawn, for DNA to be recovered.

Lambda Vector Types

Lambda Replacement Vectors

• These vectors contain have restriction sites for phage propagation.
• The lambda genome is removed and replaced by foreign DNA.
• Ligation happens in ratio of arms to target DNA to create concatemers with replication complexes and forks of interspersed vector and target molecules.
• Examples include EMBL4, EMBL3, and λ DASH.

Lambda Insertion Vectors

• Vectors cloned into an insertion vectors cleave phage DNA with a restriction site that cuts but once.
• Due to no phage DNA being removed, insertion vectors result in smaller sized target DNA inserts.
• Vector Phage λ gt 10 is commonly used.

Cosmids

• Cosmid contains a phage sequence so it can can be packaged and transmitted to form like a phage vector.
• Cosmid composition is (cos site (lambda phage) + plasmid (pBR322) , the resultant cosmids are ~5.4kbp in size.
• A cosmid, pJB8, contains:
  • An origin of replication (ori) to replicate as a bacterial plasmid.
  • An ampicillin resistance gene (amp) as a selectable marker
  • A cos gene for packing phage DNA into protein coats
  • Restriction sites for cloning, such as BamHI, EcoRI, ClaI, and HindIII.
• Cosmids can carry inserts of up to 50 kb of DNA.
• The recombinant cosmid is packaged into a lambda phage head to form an infective phage particle, delivers rDNA into E. coli, and infects the cell.

Phagemid

• A phagemid is a hybrid of a plasmid and a filamentous coliphage (M13) with a size of 1500bp and a ~44 kb insert size.
• M13 phagemids contain single-stranded circular DNA.
• Bacteriophage infection of E. coli results in the formation of double-stranded DNA replicative intermediates.
• Single-stranded DNA then packaged into a virion with replication origins of the plasmid and coliphage incorporated.
• Components of phagemid:
  • Has an origin of replication (ORI) from f1 phage (ssDNA type).
  • Contains an antibiotic resistance gene as a selectable marker (AmpR).
  • Has multiple cloning sites.
  • Has the Lac Z gene for blue-white screening.
  • Contains an origin of replication from a plasmid.
  • Has a lac promoter.
  • Carries a copy of the gene that encodes either gIIIp or gVIIIp peptides/proteins.
• Production of phages containing any phagemid genomes is achieved only when there are additional phage-derived proteins added.
• Helper phages with there own origins of replication are superinfected cells and contain packaging singals.
• Ex-phages include, CT-Phage and Hyper-phage.

Advantages of Phagemids

• Carrying capacity is larger than that of phage vectors
• Show a higher efficiency in transformation
• Can be exploited to generate copies of single stranded DNA template for sequencing purposes

Phagemid Construction

• Cloning is done in multiple sites in the double stranded circular DNA of any vector, introduced into E.coli by transformation
• Synthesis of single stranded DNA from the phage fl origin is induced by superinfection with a helper phage
• The phagemid is packaged and secreted from any bacterium when co-infected.
• Bacteria secreted from the phagemid also contain the gene of interest.

M13-Based Single-Strand Vectors

• M13 filamentous bacteriophage of E. coli is 870 nm long and 6 nm wide and has a protein coat (capsid), made of capsomeres.
• This filamentous single-stranded DNA phage infects a bacterial cell by adsorbing it and then enters through a pilus.
• The M13 genome has the following characteristics:
  • Circular single-stranded DNA.
  • 6400 base pairs long.
  • Codes for 10 genes, including
  • Gene VIII, which codes for the major structural protein.
  • Gene III, which codes for the minor coat protein.

Development of M13 into a Cloning Vector

• Introduction of the lacZ' gene into the intergenic sequence gave rise to M13 mp1, which forms blue plaques on X-gal agar
• Polylinkers were inserted into the lacZ' to create M13 vectors with cloning sites.
• The lac Z gene does not have any specific restriction sites.
• The hexanucleotide sequence GGATTC can be converted into the sequence GAATTC, becoming an ECO R1 site, which is called the M13 mp2.
• M13 mp2 lacks any sticky ends.
• If a foreign DNA is inserted into the unique Eco RI site of M13 mp2, this will cause a insertional inactivation of lac Z
• M13 mp7 is a derivative that will yield a M13 mp7 is created when a polylinker is inserted into the Eco RI site of the lac Z gene.
• The polylinker is designed in such a way that it does not inactive the lac Z gene.
• When a vector phage M13 cuts w/Eco RI, Bam HI, Sal I or Pst I, the polylinker will excised to form sticky ends recombinant M13 mp7 with 4 insertional sites.