Transforming E. coli with a Recombinant

Plasmid

What is biotechnology?

Employs use of living organisms in technology and medicineModifying living organisms

for our useMass production

Recombinant DNARecombine DNA fragments

from various sources

IntroductionRestriction enzymes

Cut DNA molecules from various organisms and recombine pieces Recombinant DNA

Restrict the growth of viruses in bacteria Digest the DNA molecule at specific nucleotide sequences Restriction fragments

DNA fragments Sticky ends

Allow annealing and recombination of DNA fragments from different sources

Engineering the Plasmid: ligation of rfp gene into p-ARA

sticky endBamH I

sticky endHind III

sticky endBamH I

sticky endHind III

Bruce Wallace

Introduction

Bacterial plasmids Circular pieces nonessential

DNA found in bacteria Can be engineered to carry genes

Express proteins encoded by these genes

pGLO Plasmid

Contains:Green Fluorescent Protein gene

Codes for green fluorescent protein

Ampicillin resistance gene

Great example of :

DNA RNA PROTEIN TRAIT

Introduction

Purpose:Get the pGLO plasmid containing the gfp gene into

bacterial cellsGet those cells to express the gfp gene and make the

mutant fluorescent protein

TransformationProcess of taking up foreign pieces of DNA In this case, a plasmid

Not very common in mature! Can occur under experimental conditions (1 cell in a

thousand!)

Plasmid DNA Insertion

Transgenic Colony Allowed to Grow

Introduction

What are competent cells?!?!Cells ready to receive plasmids through lab proceduresSoaked in calcium chloride

PM and DNA negatively charged Calcium ions (Ca++) neutralize charges to allow for

plasmid to pass through PM

Need to “heat shock” cellsCreates pressure differences to allow cell membrane to

be permeable to DNACold then hotThen feed and recover!!

MediaSpread cells on various plates (3)

LB plate Only bacterial food “control”

LB/amp plate Contains ampicillin

Antibiotic prevents bacteria from forming CW Cells that contain ampr produces protein that destroys

ampicillin “-” and “+”

LB/amp/ara plate Contains arabinose

Needed to express the gfp gene If take up plasmid, this helps with transcription of gene

Predictions……

LB LB/amp LB/amp/ara

-pGLO-pGLO +pGLO

+pGLO

LB/amp

Predictions……

LB LB/amp LB/amp/ara

-pGLO-pGLO +pGLO

+ +- ++pGLO

LB/amp