pGLO Transformation

Transforming Genes Genetic transformation is a method of inserting a gene into an organism in order to change the trait(s) of the organism. This is used in agriculture (genes for frost resistance etc), Bioremediation (bacteria used to digest oil spills) and Gene Therapy (a disease causing gene replaced with healthy one).

The GFP gene. You will be transforming bacteria with a gene that codes for ‘’Green Fluorescent Protein’’ Real life source of this protein is the Bioluminescent Jellyfish ‘’Aequorea Victoria’’ http://www.youtube.com/watch?v=jCKKxi_bhdY

Bioluminescence Other organisms in nature display bioluminescence: http://www.youtube.com/watch?v=ku5OsM9mk9Q

GFP gene Scientists have successfully transformed the GFP gene into a variety of organisms: http://www.youtube.com/watch?v=NfEIp2UJC6k http://www.youtube.com/watch?v=fYN1KzFdjlw

GFP At the end of this experiment, the bacteria will express their newly acquired GFP gene and will glow brilliant green under UV light. The gene will be transformed into the bacteria with the use of a ‘’plasmid’’.

Plasmids Bacteria have DNA contained in one chromosome. They also have small pieces of Circular DNA called plasmids. Plasmids contain genes which are beneficial to the bacteria’s survival. In nature, different bacteria can exchange plasmids back and forth in order to ‘’share’’ beneficial traits.

Bio-Rad’s pGLO plasmid. This plasmid encodes for the GFP gene and a gene for ampicillin resistance. It also contains a gene regulation system.

The Gene Regulation System The Arabinose Operon An operon is a set of genes that work together. The arabinose operon uses both positive and negative control.

The Arabinose Operon The operon contains three different genes (ara A, ara B, and ara D) that code for three enzymes needed to convert arabinose to a usable form. A fourth gene codes for a protein (ara C) that acts to regulate the structural genes. ara A, ara B, ara D Regulatory Gene Activator binding site (Structural Genes) Promoter

The Arabinose Operon Without Arabinose. Ara C (=protein) binds to activator binding site. This causes transcription of structural genes (ara A,B,D) = positive control It also prevents transcription of ara C (as it is no longer needed.) = negative control. When Ara C binds to other sites, it inhibits the structural genes. These genes are not usually active. Ara C ara A, ara B, ara D Regulatory Gene Activator binding site (Structural Genes) Promoter

The Arabinose Operon With Arabinose. binds with the arabinose and changes shape this promotes the attachment of RNA polymerase, leading to transcription Arabinose Transcription Ara C ara A, ara B, ara D Regulatory Gene Activator binding site Promoter (Structural Genes)

Bacteria Bacteria are used in this genetic transformation because: They are single celled. (so accessing the DNA is easier) They reproduce very quickly so the results of passing on genes is seen very quickly.

Step 1: Observing E-Coli Colonies In order to make observations later on about whether the genetic transformations have taken place, the first stage is to observe ‘normal’ e-coli colonies. Consider the following: Number of colonies Size of: 1) the largest colony 2) The smallest colony 3) The majority of colonies Colour of the colonies Distribution of the colonies on the plate Visible appearance when viewed with ultraviolet (UV) light The ability of the cells to live and reproduce in the presence of an antibiotic such as ampicillin

Introducing the pGLO plasmid into E-Coli cells. To move the pGLO plasmid DNA through the cell membrane you will: 1. Use a transformation solution of CaCl2 (calcium chloride) 2. Carry out a procedure referred to as heat shock For transformed cells to grow in the presence of ampicillin you must: 3. Provide them with nutrients and a short incubation period to begin expressing their newly acquired genes

Now carry out the procedure Follow the stages in the booklet When you have completed the practical, fill in the answers to the questions on page 10.