transforming e. coli with a recombinant plasmid. what is biotechnology? employs use of living...
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Transforming E. coli with a Recombinant
Plasmid
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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
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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
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Engineering the Plasmid: ligation of rfp gene into p-ARA
sticky endBamH I
sticky endHind III
sticky endBamH I
sticky endHind III
Bruce Wallace
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Introduction
Bacterial plasmids Circular pieces nonessential
DNA found in bacteria Can be engineered to carry genes
Express proteins encoded by these genes
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pGLO Plasmid
Contains:Green Fluorescent Protein gene
Codes for green fluorescent protein
Ampicillin resistance gene
Great example of :
DNA RNA PROTEIN TRAIT
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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!)
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Plasmid DNA Insertion
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Transgenic Colony Allowed to Grow
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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!!
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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
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Predictions……
LB LB/amp LB/amp/ara
-pGLO-pGLO +pGLO
+pGLO
LB/amp
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Predictions……
LB LB/amp LB/amp/ara
-pGLO-pGLO +pGLO
+ +- ++pGLO
LB/amp