Transcript Bacterial Transformation with (pGLO Plasmid)

Bacterial Transformation with
(pGLO Plasmid)
Lab #9: Molecular Biology
Purpose of this Lab
• Learn how to insert a gene into bacteria
(Heat Shock)
• Analyze how a gene can transform an organism and
express that gene
• Provide evidence that bacteria can take in foreign
DNA in the form of a plasmid
• Reinforce the following process:
DNA  RNA  Protein  Trait
• Observe how genes are regulated
Applications of Genetic Transformation
• Used in many areas of Biotechnology
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Agriculture (pests, frost, & drought)
Bacteria (oil spills)
Gene therapy (sick cells into healthy cells)
Medicine (produce insulin & hormones)
Key Terms to Know
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DNA:
Bacteria:
Growth media:
Ampicillin:
Arabinose:
Heat shock
• GFP:
Plasmid
E. coli (strain: HB101K-12)
LB Broth (Luria & Bertani)
Antibiotic kills bacteria “amp”
Sugar source for energy & carbon
Process that increases permeability
of the cell membrane to DNA
Green Fluorescent Protein (w/UV)
The Genes of Interest
• Ampicillin resistance
• Gene regulation proteins-activate the GFP gene
when arabinose is present
• GFP: Green Fluorescent Protein
-originally isolated from the jellyfish:
Aequorea victoria
Supplies for each Group of 4-5
(1) E. Coli-starter plate: has colonies present “LB” S
(4) Agar plates: 1-LB
2-LB/amp 1-LB/amp/ara
(1) Styrofoam holder tray w/ four vials
(5) Pipets
(7) Yellow inoculation loops: 1 package
(1) Cup of crushed ice & water
(2) Sharpie marking pen
Masking tape
Check your Materials-See List
• Take out what you’ll need
***(leave packages sealed)
• Label your capped vials as follows:
– Yellow:
“ - pGlo”
– Orange:
“ + pGlo”
– Green:
“TS”- Transformation Soln.
– Pink:
“LB broth”
Using the Pipet Properly
• Find the markings for each graduated volume on the pipet
Basic Process
-Begin with “Starter” colonies of E. coli
-Place a colony into each of the two tubes provided.
labeled:
-pGlo and +pGlo
(yellow loop)
-Add the pGlo plasmid to the +pGlo tube only
(yellow loop)
-Place tubes on ice (10 min)
bottom of tubes should be exposed to the ice
-Label the four plates as indicated in your guide
-Heat Shock the tubes in water bath (50 sec.)
-Return to the ice (2 min)
-Add 250 L of LB broth to both tubes (sterile pipette each time)
-Add 100 L of tube contents to the appropriate plates
-Spread the samples (w/yellow loop) & tape all four plates together. Be
sure your period and group name is clearly indicated.
Adding Transformation Solution
Transferring Colonies, Labeling the
Plates, & Using Heat Shock
The Process of Heat Shock
• Helps to increase the bacterial uptake of foreign
DNA
• Membrane becomes more permeable to DNA
• Time is essential:
-ice  water bath (42ºC) for 50 sec. ice
• The number of transformants should increase
Adding the Bacteria to the labeled
Plates
Expected Results
PLATES
+pGlo
LB/amp
+pGlo
LB/amp/ara
OBSERVATIONS
Many colonies with white appearance
Transformation observed (resistance to amp)
NO fluorescence (No arabinose present)
Many transformed white colonies
Fluoresce bright green under UV light
-pGlo
LB/amp
(CONTROL)
No Bacterial growth present on the plate
No transformation
-pGlo
LB only
(CONTROL
Bacteria present with whitish colonies
(regeneration of the starter plate)