pGLO Notes Biology III Spring 2012

Introduction to Transformation  Gene: piece of DNA which provides the instructions for making a protein  Genetic Transformation: change caused by genes.  Changes the organism’s traits  Caused by the insertion of genes from different organisms (both same and different species)

Bacterial Transformation Plasmids Chromosomal DNA Bacterial Cell The uptake of DNA

PLASMID Extrachromosomal DNA Often carry genes for antibiotic resistance Can be passed from one bacterium to another

Central Framework of Molecular Biology DNA RNA ProteinTrait

pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)

pGLO Transformation Lab  For this lab:  Transform bacteria with a gene that codes for Green Fluorescent Protein (GFP).  Source: Bioluminescent jellyfish Aequorea Victoria  Causes jellyfish to glow in the dark under ultra violet light

Links to Real-world  GFP is a visual marker  Study of biological processes (example: synthesis of proteins)  Localization and regulation of gene expression  Cell movement  Cell fate during development  Formation of different organs  Screenable marker to identify transgenic organisms

Using GFP as a biological tracer

About the lab…  Learn about the process of moving genes from one organism to another.  Transfer plasmids containing the GFP gene into Ecoli.  Bio-Rad’s unique pGLO plasmid encodes the gene for the GFP and a gene for resistance to antibiotic ampicillin  Contains a gene regulation system

Gene Regulation  Organisms regulate expression of their genes and proteins present in the cells  Allows for adaptation to the environment  Eliminate wasteful protein production  Ex. Ecoli and Catabolism (breaking down) of arabinose (sugar)  Genes involved in breaking down sugar are not expressed when no sugar is present  How is this so???

Regulation of Genes  Regulation of the expression of proteins:  Occurs at the level of transcription from DNA into RNA  Takes place at a promoter  Promoter=where transcription begins

pGLO plasmid bla (beta-lactamase) - On all time - Makes protein that breaks down ampicillin - Provides ampicillin resistance GFP-Green Fluorescent Protein - Glows green in fluorescent light ARABINOSE OPERON (INDUCIBLE) Turns on when arabinose sugar is present Allows bacteria to digest this sugar pGLO ori bla GFP araC Ori- Plasmid Replication genes

Ecoli Plasmid ori araC araD araA araB Arabinose

Specialized Genes Operator = "on/off" switch for operon Regulator = makes repressors to turn off an entire operon Repressor = Binds to operator, turn off gene expression Inducer = Joins with an active repressor, inactivates it Co-repressor = Joins with inactive repressor, converts it to active

Operon Complex RNA Polymerase must bind to the promoter site and continue past the operator site to transcribe mRNA

Arabinose Operon RNA Polymerase BAD araC Effector (Arabinose) araC BAD Promotor (P BAD ) DNA binding Protein: Represses Transcription Genes coding for digestive enzymes BAD araC Transcription

Ara-GFP Operon RNA Polymerase araC GFP Gene araC GFP Gene araC GFP Gene Effector (Arabinose) Promotor (P BAD ) Transcription  GFP only produced in the presence of Arabinose  Genes coding for digestive enzymes have been replaced by the GFP gene: no metabolism of arabinose

How Does it GLOW? Unique 3-D Structure of GFP Resonates when exposed to ultraviolet light Gives off energy in the form of green fluorescent light Learn more about GFP:

How does it work? GFP Beta lactamase (ampicillin resistance) pGlo Plasmids Bacterial chromosomal DNA Cell wall Transform bacteria with the pGlo plasmid and grow under various conditions

Methods of transformation DNA molecules are too large to easily diffuse or be transported though the cell membrane Electroporation Electrical shock makes cell membranes permeable to DNA Calcium Chloride/Heat Shock Chemically-competent cells uptake DNA after heat shock

Electroporation

Transformation

Transformation Procedure: Overview Suspend bacterial colonies in Transformation Solution Add pGLO plasmid DNA Place tubes on ice Heat shock at 42 o C and place on ice Incubate with LB nutrient broth Streak plates

Why perform each step?  CaCl 2 treatment on ice crytallizes fluid membranes and stabilizes distribution of charged molecules  CaCl 2 Transformation solution provides Ca ++ cations that neutralize the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane, allowing the DNA to enter the cells Ca ++ O CH 2 O P O O O Base CH 2 O P O O O Base OH Sugar O Ca ++

Picture, Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Why perform each step? HHeat-shock increases permeability of cell membrane LLuria-Bertani Nutrient broth incubation allows beta lactamase expression Beta lactamase (ampicillin resistance) pGlo Plasmids Bacterial chromosomal DNA Cell wall

Gene selection Grow transformed bacteria and control bacteria under various conditions. On which plates will colonies grow? Which colonies will glow?

The pGlo System Timing is important…be efficient!! Mix contents before pipetting!!! A film of plasmid must be on the loop!

Sterile Technique Bacteria are UBIQUITOUS…they are found EVERYWHERE! Sterile technique refers to procedures that reduce the possibility of contamination…these techniques protect YOU, your CULTURES and REAGENTS, and LAB EQUIPMENT

pGlo Lab Considerations Teacher Considerations Work surfaces Hands Glassware Agar Petri plates Inoculation loops Solutions/Cultures Pipettes Student Considerations  Work surfaces  Hands  E.coli starter plates  Assorted agar plates  Inoculation loops  Solutions/Test tubes  Pipettes

Closing Considerations ALWAYS decontaminate you work surfaces with a disinfecting solution: 20ml of liquid household bleach in 1L of tap water. Spray the solution on work surfaces and let stand for 2 minutes and wipe away ALWAYS thoroughly scrub hands for at least 20 seconds with soap and hot water before leaving the lab area

Volume Measurement