Virtual Cloning Tutorial - Introduction

Glowing proteins - a guiding star for biochemistry

The remarkable brightly glowing green fluorescent protein, GFP, was first observed in the beautiful jellyfish, Aequorea victoria in 1962. Since then, this protein has become one of the most important tools used in contemporary bioscience. With the aid of GFP, researchers have developed ways to watch processes that were previously invisible, such as the development of nerve cells in the brain or how cancer cells spread.


Tens of thousands of different proteins reside in a living organism, controlling important chemical processes in minute detail. If this protein machinery malfunctions, illness and disease often follow. That is why it has been imperative for bioscience to map the role of different proteins in the body.

The 2008 Nobel Prize in Chemistry rewards the initial discovery of GFP and a series of important developments which have led to its use as a tagging tool in bioscience. By using DNA technology, researchers can now connect GFP to other interesting, but otherwise invisible, proteins. This glowing marker allows them to watch the movements, positions and interactions of the tagged proteins.

Researchers can also follow the fate of various cells with the help of GFP: nerve cell damage during Alzheimer's Disease or how insulin-producing beta cells are created in the pancreas of a growing embryo. In one spectacular experiment, researchers succeeded in tagging different nerve cells in the brain of a mouse with a kaleidoscope of colours.

Once a fusion protein is made, it can be visualised in live cells by microscopy techniques as shown in this video.

In this tutorial, we will attempt to make a functional GFP fusion protein. The process of cloning involves an insert and a vector. Our insert is the Human Proinsulin gene and our vector is a plasmid named pEF-F3-EGFP which encodes an enhanced copy of the GFP gene (EGFP). Using the cloning tools in Geneious, we can conduct an in-silico experiment to investigate how best to make a plasmid with a functional fusion protein.


Exercise 1: Viewing 3D structure of GFP
Exercise 2: Cloning Vector
Exercise 3: Human Proinsulin
Exercise 4: Cloning Primer Design
Exercise 5: Introduce Cut Site using PCR
Exercise 6: Insert into Vector