7.1 Transferring of a gene between different organisms

7.1.1 Transferring human insulin gene into bacteria.

Introduction

Diabetes is a condition that is affecting increasing number of people each year. Previously, diabetes was treated by injecting insulin from animal sources such as pigs and cows. However, scarcity in pancreas of pigs and cows restricts supply of insulin and makes it more costly. Furthermore, allergies resulting in use of insulin from animal sources have led to deaths. There was also worry about infecting of animal diseases through the use of insulin from animal sources.

Insulin produced by E.coli has several advantages:

1. E.coli is a small organism and it can be produced in large scale using fermenters. Supply of insulin is greatly increased and the cost of production is greatly reduced.

2. Insulin from E.coli is more identical to natural insulin and thus it does not induce allergic response from users.

3. Risk of contracting disease from the use of insulin produced by E.coli is low.

7.1.1.1 Formation of recombinant plasmid

• Human insulin is a protein consisting of 51 amino acids. The human insulin gene is located on Chromosome 11.

1 A specific restriction enzyme (eg. EcoRI) locates specific DNA sequences on the DNA segment with the human insulin gene - G|AATTC

CTTAA|G.

The restriction enzymes will cleave at the regions shown using straight lines, generating two sticky ends.

2 The same restriction enzyme is used to digest the plasmid to generate an open plasmid with two sticky ends.

3 The sticky ends of the open plasmid are complementary to the sticky ends of the human insulin gene. DNA ligase is then used to join the sticky ends. The resulting closed plasmid with the gene of interest (ie human insulin gene) is known as the recombinant plasmid.

7.1.1.2 Bacterial Transformation

The recombinant plasmid is introduced into the recipient organism, ie. E.coli by heat shock process. Heat shock process refers to the treatment of E.coli cells and recombinant plasmid with alternating high and low temperatures to introduce temporary holes on E.coli cell wall and cell surface membrane to allow recombinant plasmid to enter.

E.coli with the recombinant plasmid is considered to be transgenic and they are cultured in large fermenters to mass produce insulin proteins.

7.1.2 Transferring Bt toxin from one bacteria into crop plants

7.1.2.1 Introduction

Bacillus thuringiensis is a soil bacterium that produces a protein toxin known as Bt toxin, that is toxic to insects. Bt toxin is produced in an inactive, crystalline form. When insects ingest Bt toxin, the toxin is converted to its active, toxic form that destroys the insect.

7.1.2.1 Formation of recombinant plasmid

1. A specific restriction enzyme is used to cut the Bt gene from the Bacillus thuringiensis DNA to form two sticky ends.

2. A suitable plasmid is cut using the same restriction enzyme to form an open end with two sticky ends.

. The sticky ends of Bt gene will bind to sticky ends of the open plasmid by complementary base pairing. DNA ligase is used to join the sticky ends to form a recombinant plasmid.

7.1.2.2 Transformation

Method 1: Minute gold particles coated with the recombinant plasmid are loaded onto a gene gun. The gene gun injects the minute gold particles coated with the recombinant plasmid into the crop plant cells.

Method 2: The recombinant plasmid is inserted into a bacteria that can infect plants known as Agrobacterium. Agrobacterium containing the recombinant plasmid will then infect crop plants and transfer the recombinant plasmid to the crop plants.

7.1.2.3 Advantages of genetic engineering Bt-proteins into crop plants

1. As fewer crop plants are consumed by pests, crop production will increase.

2. Less pesticides need to be applied, reducing air and water pollution.

7.1.2.4 Disadvantages of genetic engineering Bt-proteins into crop plants

1. Useful insects may be killed.

2. Insect pests may develop resistance to the poison produced by the plant.

3. Removal of insects may cause ecological food chains to be disrupted and this affects the ecological balance.