E-Learning Course: Recombinant DNA Biology

Course Code: MSLSC2003C04Course Coordinator: Dr. Tara Kashav

Topic: DNA Cloning Vectors

Note: All the material is compiled/modified from textbooks/freely available e-resources just meant for learning

purpose of students

Cloning vectors

• Plasmids

• Virus based vectors

• Bacterial Artificial Chromosomes

• Yeast Artificial Chromosomes

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Forms of plasmid

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Plasmid propertiesuseful to molecular biologists

• Small circular DNA molecule – Replicates separately from host chromosome– Range in size from 5,000 to 4,00,000 bp

• To survive in the host cell– use cell’s resources for their own replication and gene

expression• Naturally occurring plasmids

– usually have a symbiotic role in the cell– May provide genes that confer resistance to antibiotics– Perform new functions for the cell

• Ex: Ti plasmid of Agrobacterium tumefaciens allows the host bacterium– To colonize the cells of plants– Make use of the plant’s resources

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E. coli plasmid pBR322• Constructed in 1977, in lab of Herbert Boyer at University of

California

– It was named after Francisco Bolivar Zapata, postdoctoral researcher.

– p stands for "plasmid," and BR for "Bolivar" and "Rodriguez.“

• consists of

• origin of replication, or ori

– Initiation of Replication by cellular enzymes

– Helps to propagate the plasmid

– Has an associated regulatory system to maintain pBR322 at a level of 10 to 20 copies per cell

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E. coli plasmid pBR322

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E. coli plasmid pBR322

• Small size of plasmid (4,361 bp) facilitates

– its entry into cells

– the biochemical manipulation of the DNA

• Size is generated by trimming away many DNA segments from a larger, parent plasmid

– Seq that the molecular biologist does not need !!!

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• Genes that confer antibiotic resistance

– Tetracycline (TetR) : Selection of cells with intact plasmid

– Ampicillin (AmpR): selection of recombinant plasmid

• Unique R.E. recognition seq

– PstI, EcoRI, BamHI, SalI, and PvuII

– Sites where the plasmid can be cut to insert foreign DNA

E. coli plasmid pBR322

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Effect of EtBr intercalation

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Conjugated Non-Conjugated plasmids

Contains tra genes Lacks transfer genes (tra)

Promotes sexual conjugation b/w different cells: can be transferred from one bacterium to another via sex pili

No transfer possible until associated with conjugative plasmids

Colicin E1 production

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Origin of pBR322

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pBR322

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Plasmid copy number• Depending on the origin used

– Plasmid copy number may vary from 1-100-1000/cell

– Provides many options for investigators

• Relaxed origin of replication

– Plasmid replication is not tightly linked to cell division

– Plasmid replication initiated more frequently than chromosomal replication

– large number of plasmids will be produced per cell

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Co-expression of Plasmids• When a researcher wants to introduce two or more diff

plasmids into a bacterial cell

– Each plasmid must have a different replication origin

– Same origin of replication leads to incompatibility as

– Regulation of one will interfere with replication of other

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E.coli Promoter

Four structural domains can be recognized

• position 1, the purine initiation nucleotide from

• which RNA synthesis begins;

• position −6 to −12, the Pribnow box;

• region around base pair −35;

• the sequence between base pairs −12 and −35.

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pUC 18 vectorproduced at the University of California

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1.PCR amplification

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Bacterial transformation

• Key step in molecular cloning

• Goal - to produce multiple copies of a recombinant DNA

• DNA plasmid is introduced into competent strain of bacteria

• Bacteria then replicate sequence of interest

• Four key steps are:

– Preparation of competent cells

– Transformation

– Cell recovery period

– Cell plating

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protocol

• a single fresh colony of desired strain is taken from agar plate

• Inoculated into liquid medium for a starter culture

• From starter culture, secondary culture initiated

• Secondary culture monitored for active growth

– By measuring optical density at 600 nm (OD600)

• To obtain high transformation efficiency

– Cell growth be in the mid-log phase at the time of harvest

– OD600 between 0.4 - 0.6

– sterile labware, media, and reagents

– Once cells are harvested all samples, kept at 0–4°C

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Screenable marker- β-galactosidaseBlue-White selection

• pUC plasmid used to transform the host cell E. coli

– the gene β-gal may be switched on by adding inducer IPTG (isopropyl- β -D-thiogalactopyranoside)

• Enzyme β-galactosidase is produced

– Hydrolyse a colourless substance called X-gal (5-bromo-4-chloro-3-indolylb-galactopyranoside) into a blue insoluble material (5,5’-dibromo-4,4’–dichloroindigo)

• In recombinant plasmid: Insertional inactivation

– a non-functional enzyme is unable to carry out hydrolysis of X-gal: NOT blue !!

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BLUE-WHITE selection

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Limitations of plasmid as vector• Inserts greater than 5 kb increase plasmid size

– Transformation of bacterial cells becomes less successful

• Difficult to clone DNA segments longer than about 15 kp

Virus based vectors• To propagate larger fragments of DNA in bacteria

– bacteriophages have been adapted as vectors

– commonly derived from λ bacteriophage

– 16-fold more cloning efficiency as compared to plasmid cloning vectors

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Maximum DNA insert possible with different cloning vectors

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