MB 206 : Module 2 - B

1. MB 206 : Module 2 - B Extraction of Nucleic Acids (Genomic DNA, mRNA and Plasmid DNA)

2. Now, what is a plasmid ? Let us restart with our current Understanding of Plasmids 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). Sizes: 1 to over 400 kb. Copy numbers: 1 - hundreds in a single cell, or even thousands of copies. 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). Schematic drawing of a bacterium with its plasmids. (1) Chromosomal DNA. (2) Plasmids

3. Types of Bacterial Plasmids 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. Based on their function,there are five main classes: Col-plasmids: contain genes that code forcolicines, 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. Plasmids can belong to more than one of these functional groups.

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

5. Development of Plasmid Vectors Plasmids serve as important tools in genetics and biochemistry labs, where they are commonly used to multiply or express particular genes. Plasmids used in genetic engineering are called vectors. Vectors are vehicles to transfer genes from one organism to another and typically contain a genetic marker conferring a phenotype. Most also contain a polylinker or multiple cloning site (MCS), with several commonly used restriction sites allowing easy insertion of DNA fragments at this location. Many plasmid vectors are commercially available. Old vector pBR322: 4.36kb, Ampicilin-R, Tetracylin-R, 15-20 copies/cell Old vectors pUC18/19: 2.69kb, Ampicilin-R, LacZ operon, 500-700 copies Stratagen pBS-KS: 3.0kb, Ampicilin-R, LacZ operon, 500-700 copies/cell Promega pGEM-T: 3.0 kb, Ampicilin-R, LacZ operon, 500-700 copies/cell Invitrogen TOPO-TA: 3.96kb, Ampicilin-R, Kan-R, LacZ, 500-700 copies pCAMBIA vectors: >10kb, Amp-R/Kan-R/Hyg-R, LacZ, 1-3 copies see more at http://seq.yeastgenome.org/vectordb/vector_pages/

6. MCS Plasmid Vectors

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

8. Green Algae for antibody production Transgenic Arabidopsis expressing GFP to study PDI functions Application of Plasmid Vectors In Pharmaceutical and Agriculture Bioengineering 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.

9. (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 Plasmid Isolation from Bacteria How to rapidly isolate plasmid?

10. Midi Prep Mini Prep spectrophotometer and gel electrophoresis Plasmid DNA Isolation continued Tranditional Ways (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).

11. 1. Isolation of plasmid DNA • Plasmid DNA can be isolated from bacterial cells using alkaline/SDS based mini-plasmid purification method. • The method of isolating plasmid DNA using alkaline lysis with SDS was first reported by Birnboim HC and Doly J. in 1979 (Nucleic Acids Res. 1979 Nov 24;7(6):1513-23). “A rapid alkaline extraction procedure for screening recombinant plasmid DNA”.

12. 1. Isolation of plasmid DNA • This method has been used for more than 25 years to isolate plasmid DNA from E. coli. • It is very simple and permits the analysis of 100 or more clones per day by gel electrophoresis. • The plasmid DNA is pure enough to be digestible by restriction enzymes.

13. 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.

14. CH3 | CH2 | CH2 | CH2 | CH2 | CH2 | CH2 | CH2 | CH2 | CH2 | CH2 | CH2 | O | S | O Bacterial chromosome Plasmid Please go to the previous step to replay the animation. Click to start the animation • SDS is a strong ionic detergent with a negative charge. • At high pH,SDScan open the bacterial cell wall, denature chromosomal DNA and proteins, and release plasmid DNA into the supernatant. o o Sodium dodecyl sulfate (SDS)

15. Click on animation to start playing

16. 3’ 5’ C G T A T A T A T A C G C G T A C G T A 5’ 3’ • 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. Circular dsDNA dsDNA in supercircular form Hydrogen-bonded base pairs