Download Document

Transforming E. coli with
a Recombinant Plasmid
What is biotechnology?
 Employs use of living organisms
in technology and medicine
 Modifying living organisms for
our use
 Mass production
 Recombinant DNA
 Recombine DNA fragments from
various sources
Introduction
 Restriction 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
Bruce Wallace
BamH I
sticky end
Hind III
sticky end
Hind III
sticky end
BamH I
sticky end
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
cells
 Get those cells to express the gfp gene and make the mutant
fluorescent protein
 Transformation
 Process 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 procedures
 Soaked 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” cells
 Creates pressure differences to allow cell membrane to be
permeable to DNA
 Cold then hot
 Then feed and recover!!
Media
 Spread 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……
-pGLO
LB
-pGLO
+pGLO
LB/amp
LB/amp
+pGLO
LB/amp/ara
Predictions……
-pGLO
+pGLO
-pGLO
+pGLO
+
-
+
+
LB
LB/amp
LB/amp
LB/amp/ara