2. Plasmid is autonomously replicating, extrachromosomal circular DNA molecules, distinct from the normal chromosomal DNAs and nonessential for cell survival under nonselective conditions. Episome no longer in use. They usually occur in bacteria, sometimes in eukaryotic organisms (e.g., the 2-um-ring in yeast S. cerevisiae).bacteriaeukaryotic organismsS. cerevisiae Sizes: 1 to over 400 kb. Copy numbers: 1 - hundreds in a single cell, or even thousands of copies.cell Every plasmid contains at least one DNA sequence that serves as an origin of replication or ori (a starting point for DNA replication, independently from the chromosomal DNA).origin of replicationDNA replication Let us restart with our current Understanding of Plasmids Schematic drawing of a bacterium with its plasmids. (1) Chromosomal DNA. (2) Plasmids Now, what is a plasmid ?

3. Fertility-(F)plasmids: they are capable of conjugation or mating. Resistance-(R) plasmids: containing antibiotic or drug resistant gene(s). Also known as R-factors, before the nature of plasmids was understood.conjugation Types of Bacterial Plasmids Col-plasmids: contain genes that code for colicines, proteins that can kill other bacteria. Degrative plasmids: enable digestion of unusual substances, e.g., toluene or salicylic acid. Virulence plasmids: turn the bacterium into a pathogen.colicinesproteinstoluene salicylic acidpathogen Plasmids can belong to more than one of these functional groups. Based on their function, there are five main classes:

4. Antibiotic resistance R-plasmids often contain genes that confer a selective advantage to the bacterium hosts, e.g., the ability to make the bacterium antibiotic resistant.genesantibiotic resistant Some common antibiotic genes in plasmids: amp r, APH3’-II (kanamycin), tet R (tetracycline),cat R (Chloramphenicol), spec r (spectinomycin or streptomycin), hyg r (hygromycin). Some antibiotics inhibit cell wall synthesis and others bind to ribosomes to inhibit protein synthesis ori Amp-R Schematic drawing of a plasmid with antibiotic resistances Kan-R

5. Plasmid Vectors MCS

6. Application of Plasmid Vectors How it works? (a)Initially, the gene to be replicated is inserted in a plasmid or vector. (b)The plasmids are next inserted into bacteria by a process called transformation. transformation (c)Bacteria are then grown on specific antibiotic(s). (d)As a result, only the bacteria with antibiotic resistance can survive and will be replicated. In Molecular Cloning

7. One of the major uses of plasmids is to make large amounts of proteins. In this case, bacteria or other types of host cells can be induced to produce large amounts of proteins from the plasmid with inserted gene, just as the bacteria produces proteins to confer antibiotic resistance. This is a cheap and easy way of mass-producing a gene or the protein — for example, insulin, antibiotics, antobodies and vaccines.insulinantibiotics Application of Plasmid Vectors Green Algae for antibody production Transgenic Arabidopsis expressing GFP to study PDI functions In Pharmaceutical and Agriculture Bioengineering

8. Plasmid Isolation from Bacteria How to rapidly isolate plasmid? (a) Inoculation and harvesting the bacteria (b) lysis of the bacteria (heat, detergents (SDS or Triton-114), alkaline(NaOH)), (c) neutralization of cell lysate and separation of cell debris (by centrifugation), Or other cell types

9. (d) collecting plasmid DNA by centrifugation (after ethanol precipitation or through filters - positively charged silicon beads), (e) check plasmid DNA yield and quality (using spectrophotometer and gel electrophoresis). Plasmid DNA Isolation continued Midi PrepMini Prep Tranditional Ways spectrophotometer and gel electrophoresis

10. Isolation of plasmid DNA About 10 years ago, this method was modified so that very pure plasmid DNA can be purified using a column specially packed with glass fiber. The plasmid DNA purified through those columns can be used directly for PCR, cloning, sequencing, in vitro transcription, synthesize of labelled hybridization probes, microinjection, electroporation and transfection, etc. 10

11. SDS is a strong ionic detergent with a negative charge. At high pH, SDS can open the bacterial cell wall, denature chromosomal DNA and proteins, and release plasmid DNA into the supernatant. 11 Bacterial chromosome Plasmid CH 3 | CH 2 | CH 2 | CH 2 | CH 2 | CH 2 | CH 2 | CH 2 | CH 2 | CH 2 | CH 2 | CH 2 | O | S | O oo Sodium dodecyl sulfate (SDS) Please go to the previous step to replay the animation.Click to start the animation

12. 12 Click on animation to start playing

13. 13 Circular dsDNA dsDNA in supercircular form 3’ C G C G C G T A T A T A T A T A T A C G 5’ 3’ Hydrogen-bonded base pairs SDS/alkaline solution can denature chromosomal DNA by disrupting the hydrogen bonds between the two complementary strands of nucleotides. However, this treatment cannot destroy the covalently closed circular plasmid DNA because they are topologically intertwined.