Experiment 1: Isolation, Cloning and Transformation of Plasmid DNA

Isolation, Cloning and Transformation of Plasmid DNA Overall Purpose of the Experiment

To transform a strain of E.coli DH5-alpha to be resistant to both ampicillin and kanamycin by creating a recombinat pUC18 plasmid by restriction digestion and cloning

Plasmid

An autonomous, self replication, extra-chromosomal DNA molecule

Cloning

Incorporating a DNA molecule into a chromosomal site or a cloning vector

Clone

A population of cells that carry a cloning vehicle with the same DNA insert

Transformation

The uptake of extra chromosomal DNA in a bacterium or yeast in which the DNA often changes the phenotype of the recipient organism

Competent cells

Cells which have been treated by chemical or physical means in order to be sensitive to foreign DNA

pUC18 plasmid

Contains ampicillin resistance gene, origin of DNA replication, portion of the lacZ gene ligated to the gene, and MCS

pKAN plasmid

Contains kanamycin resistance gene

Blue selection technique

Cells are able to make beta-galactosidase and produce blue colonies on plates containing the substrate, X-gal

White selection technique

Appearance of white colonies on the X-gal plates indicative of the presence of the kanamycin gene inserted into the pUC18 vector

What is the molecular weight of pKan plasmid

4194 bp

What is the molecular weight of the pUC18 plasmid and how many plasmid will appear on the gel image?

2,686 bp, and 2 bands will appear on the gel image

Supercoiled

Occurs when extra twists are introduced into double helix strand. Migrates faster than predicted

Nicked, Relaxed Circular Plasmid

Cellular topisomerases nick one strand of the DNA helix and relaxes the superhelical tension. Migrated the slowest in the gel

Linear Plasmid

DNA helix is cut in both strands at the same place

Circular, single stranded plasmid

DNA is permanently denatured resulting in single stranded closed circles

Restriction endonuclease digestion of pUC18 and pKAN

Open up the MCS in pUC18 using restriction enzymes that a fragment from pKAN (containing the kanamycin resistance gene) is inserted into the MCS

Ligation reaction

Ligases use ATP to link the 5' end of one DNA strand to the OH group on the 3' strand---> phosphodiester bond

Restriction endonucleases

- Palindrome sequence

- Creates sticky ends or blunt ends

- 3-letter designation nomenclature system (E.g., EcoRI, HindIII)

puC18 plasmid characteristics

1) the pMB1 replicon rep

2 ) bla gene

3) lac operon

Direct screening method

Plating on Lb/amp/kan plates and selecting for the presence of kanamycin gene

Indirect screen method

Plating on the amp/X-gal plates to detect the presence of kanamycin gene inserts and then plated onto plates containing the kanamycin gene.

LB broth

Nutriontially rich medium used to grow a variety of bacteria in the laboratory

operon

structural genes,together with promoter and operator or activator-binding sires

Promoter

A region of DNA that initiates transcription of a particular gene

Repressor

That inhibits the expression of one or more genes by binding to the operator

lac Z

Encodes beta-galactosidase, which converts lactose to allolactose (inducer of the the operon)

CAP (catabolite activator protein)

transcription activator