Recombination and Transformation (VCE SSCE Biology): Revision Notes

Recombination and Transformation

Introduction

Diabetes affects approximately 1.2 million Australians. This condition occurs when the pancreas cannot adequately produce insulin, a hormone responsible for glucose uptake from the bloodstream into cells. Whilst there is currently no cure for diabetes, the condition can be managed through regular insulin injections. This raises an important question: how can we produce enough insulin for 5% of the Australian population to use daily?

The answer lies in genetic engineering. Through bacterial transformation, scientists have developed methods to make bacteria produce human insulin and other vital proteins.

Why transform bacteria?

Bacteria are simple prokaryotic organisms that contain two types of genetic material. They have a main circular chromosome and smaller, independent loops of DNA called plasmids.

Plasmid – a small, circular loop of DNA separate from the chromosome, typically found in bacteria.

Plasmids replicate independently from the bacterial chromosome. The number of plasmids varies between individual bacteria - some may contain many plasmids whilst others have none. These plasmids can range in length up to 200,000 base pairs (200 kbp).

Scientists can edit plasmids to include target genes of interest. Once a plasmid has been modified to incorporate a foreign gene, it becomes a recombinant plasmid. When bacteria take up these recombinant plasmids from their environment through a process called bacterial transformation, they gain the ability to produce specific proteins encoded by the foreign gene.

Recombinant plasmid – a circular DNA vector that is ligated to incorporate a gene of interest.

Bacterial transformation – the process by which bacteria take up foreign DNA from their environment. Scientists use this process to introduce recombinant plasmids into bacteria.

Genetic modification – the manipulation of an organism's genetic material using biotechnology.

This technology has revolutionized modern medicine and agriculture by enabling cheaper and more efficient production methods. Bacterial transformation is now used to produce many important proteins, including:

• Insulin to manage diabetes
• Erythropoietin to treat anaemia
• Chymosin for cheese production (previously extracted from calf stomachs)
• Interferon to treat some cancers
• Growth hormone to manage growth disorders
• Hepatitis B surface antigen for vaccines
• Alpha-amylase for ethanol and high fructose corn syrup production

Making a recombinant plasmid

Creating recombinant plasmids requires genetic engineers to insert foreign DNA into bacterial plasmids. Once bacteria take up these modified plasmids, they will express the protein encoded by the foreign DNA. This process requires four key components:

1. Gene of interest
2. Plasmid vector
3. Restriction endonuclease
4. DNA ligase

Gene of interest

The gene of interest (also called the desired gene) is a DNA sequence encoding the protein scientists wish to produce.

Gene of interest – a gene scientists want to be expressed in recombinant bacteria. This gene often encodes a protein we wish to produce in commercial quantities.

The gene is typically isolated from human DNA and amplified using the polymerase chain reaction (PCR). An important feature is that bacteria can use human DNA to produce identical human proteins because the genetic code is universal. For example, a CUU codon codes for the amino acid leucine in all organisms, whether human or bacterial.

Critical requirement: Removal of introns

Introns are typically removed using two methods:

• Synthetic DNA – Genes are manufactured artificially in a laboratory using a DNA synthesizer. Introns are simply not included when the gene is created.
• Copy DNA (cDNA) – An enzyme called reverse transcriptase converts mature mRNA (which already has introns removed) back into DNA. Since the mRNA template lacks introns, the resulting cDNA also lacks introns.

Plasmid vector

A plasmid vector is a carefully designed piece of circular DNA that has been modified to be ideal for bacterial transformation experiments.

Plasmid vector – a piece of circular DNA that is modified to be an ideal vector for bacterial transformation experiments.

Vector – a means of introducing foreign DNA into an organism. Plasmids are a popular vector in bacterial transformation.

Most plasmid vectors contain four essential DNA sequences:

1. Restriction endonuclease sites

These are specific DNA sequences that can be recognized and cut by restriction enzymes. They provide locations where the gene of interest can be inserted into the plasmid. Common examples include BamHI and EcoRI restriction sites.

2. Antibiotic resistance genes

These genes allow bacteria carrying the plasmid to survive in the presence of specific antibiotics. For example:

• ampR confers resistance to ampicillin
• tetR confers resistance to tetracycline

Antibiotic resistance gene – gene which confers antibiotic resistance.

3. Origin of replication (ORI)

This is a DNA sequence that signals where DNA replication should begin in bacterial cells. It ensures the plasmid can be copied when bacteria divide.

Origin of replication (ORI) – a sequence found in prokaryotes that signals the start site of DNA replication.

4. Reporter gene

These genes produce easily observable characteristics that help identify successful genetic modification. Reporter genes have recognizable phenotypes that can be used to distinguish recombinant plasmids from non-recombinant plasmids.

Reporter gene – gene with an easily identifiable phenotype that can be used to identify whether a plasmid has taken up the gene of interest.

An example is the gfp gene, which produces green fluorescent protein that glows green under UV light.

Restriction endonuclease

Restriction endonuclease (also called restriction enzyme) – any enzyme that acts like molecular scissors to cut nucleic acid strands at specific recognition sites.

Both the gene of interest and the plasmid vector are cut using the same restriction endonuclease. This creates identical "sticky ends" on the gene and the plasmid, allowing them to join together.

For example, the restriction enzyme BamHI can be used to:

• Cut out the gene of interest from source DNA
• Create an opening in the plasmid vector (specifically within the reporter gene)

The overhanging nucleotides created by the enzyme on the gene of interest are complementary to the overhanging nucleotides on the plasmid. This complementarity allows them to form hydrogen bonds easily. Whilst blunt-end restriction enzymes can also be used, they are less targeted because any blunt end can bond with any other blunt end.

DNA ligase

Ligase – an enzyme that joins molecules, including DNA or RNA, together by catalysing the formation of phosphodiester bonds.

DNA ligase is the final component needed to create a recombinant plasmid. This enzyme joins the gene of interest to the plasmid vector by forming strong phosphodiester bonds in the sugar-phosphate backbone of DNA.

The result is a circular piece of DNA called a recombinant plasmid. However, the process is not perfectly efficient.

Transforming bacteria

Uptake of recombinant plasmids

Many bacteria can naturally take up free-floating DNA from their environment and incorporate it into their cytoplasm through transformation. Scientists exploit this natural ability to introduce recombinant plasmids into bacterial cells.

Two main methods are used to enhance plasmid uptake:

Heat shock method

Heat shock – a method that involves rapidly increasing and decreasing the temperature to increase membrane permeability in order to enhance the likelihood of bacterial transformation.

The heat shock process follows these steps:

1. Bacteria and plasmids are placed in a calcium ion solution on ice (0°C)
2. The positive calcium ions make the negatively charged bacterial membrane more permeable to negatively charged plasmid DNA
3. The solution is rapidly heated to 37-42°C for 25-45 seconds
4. The solution is immediately returned to ice (0°C)
5. The sudden temperature change increases membrane permeability, allowing plasmids to cross the membrane
6. The mixture is spread onto antibiotic-rich agar plates

Electroporation method

Electroporation – a method that involves delivering an electric shock to bacterial membranes to increase their membrane permeability and increase the likelihood of bacterial transformation.

This method is similar to heat shock but uses electricity instead of temperature:

1. Bacteria and plasmids are mixed in solution
2. An electrical current is passed through the solution
3. The electric shock makes the membrane more permeable
4. Plasmids can cross the phospholipid bilayer
5. The electric shock is stopped
6. The mixture is spread onto antibiotic-rich agar plates

Antibiotic selection

After transformation, the bacterial mixture contains both transformed bacteria (which took up plasmids) and untransformed bacteria (which did not). These need to be separated.

Each visible colony on the plate represents a successful transformation event. A single bacterium took up a plasmid, survived the antibiotic exposure, multiplied, and formed a colony of genetically identical daughter cells.

Distinguishing recombinant from non-recombinant plasmids

Transformed bacteria can contain either recombinant plasmids (with the gene of interest) or non-recombinant plasmids (which joined back to themselves). These must be distinguished from each other using the reporter gene.

Example: Using the gfp reporter gene

The gfp gene encodes green fluorescent protein, which glows green under UV light.

• Non-recombinant plasmids: The gfp gene is intact and continuous. Bacteria with these plasmids produce functional green fluorescent protein and glow green under UV light.
• Recombinant plasmids: The gfp gene is interrupted by the inserted gene of interest. The gene is non-continuous and cannot produce functional fluorescent protein. Bacteria with these plasmids do NOT glow under UV light.

Scientists can therefore identify bacteria containing recombinant plasmids by selecting colonies that do not fluoresce under UV light.

Protein production and extraction

Once bacteria containing recombinant plasmids are identified, they are:

1. Cultured to grow into large populations
2. Induced to express the gene of interest and produce the target protein
3. Harvested and processed to extract the desired protein
4. Purified to remove other bacterial proteins

The purified protein can then be used in medicine, research, or industry.

Insulin production

Insulin – a hormone secreted by the pancreas to control blood glucose levels.

Diabetes – a disease where the body cannot properly produce or respond to insulin.

Background

Before genetic engineering techniques were developed, diabetic patients received insulin extracted from the pancreases of pigs (porcine insulin) or cows (bovine insulin). These animal insulins have similar structures to human insulin, but this method had significant drawbacks:

• Required killing many animals to extract insulin from their pancreases
• Less effective at regulating blood glucose than human insulin
• Could cause immune reactions in some patients
• Expensive and inefficient

In the late 1970s, scientists successfully produced human insulin in bacteria using recombinant plasmids. This method has become the standard approach because it is significantly cheaper, more effective, and does not require animal sacrifice.

Insulin structure

Insulin has a quaternary protein structure consisting of two polypeptide chains:

• Alpha subunit (A chain)
• Beta subunit (B chain)

These two chains are joined together by disulphide bridges to form functional insulin.

The insulin production process

The complete process involves nine main steps:

Summary

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