1. Genetic Engineering and Gene Technology
define the term recombinant DNA;
explain that genetic engineering involves the extraction of genes from one organism, or the manufacture of genes, in order to place them in another organism (often of a different species) such that the receiving organism expresses the gene product
describe how sections of DNA containing a desired gene can be extracted from a donor organism using restriction enzymes;
explain how isolated DNA fragments can be placed in plasmids, with reference to the role of ligase;
state other vectors into which fragments of DNA may be incorporated;
explain how plasmids may be taken up by bacterial cells in order to produce a transgenic microorganism that can express a desired gene product;
describe the advantage to microorganisms of the capacity to take up plasmid DNA from the environment;
outline how genetic markers in plasmids can be used to identify the bacteria that have taken up a recombinant plasmid;
outline the process involved in the genetic engineering of bacteria to produce human insulin;
outline the process involved in the genetic engineering of ‘Golden Rice’
outline how animals can be genetically engineered for xenotransplantation
explain the term gene therapy;
explain the differences between somatic cell gene therapy and germ line cell gene therapy;
discuss the ethical concerns raised by the genetic manipulation of animals (including humans), plants and microorganisms

2. Genetic Engineering and Gene Technology: Key Terms
Recombinant DNA: A DNA molecule formed from DNA from 2 sources which are then joined by the enzyme DNA ligase.
Genetic engineering: branch of biotechnology characterised by the obtaining of a specific gene, placing the gene in a different organism and then causing the organism to express the gene, transcribing and translating it to create a protein. This gives the recipient a characteristic that it did not have previously.
Transgenic: an organism which contains DNA that has been added to its cells by genetic engineering.
Transformed: a transgenic organism into which the new gene has been added is descried as a transformed organism – for example, a bacteria that has taken up a DNA plasmid from the environment.
Vector: a biological structure able to successfully introduce and integrate a gene into a cell and its genome.
Annealing: the term used to describe hydrogen bond formation between complementary base pairs when sections of DNA join together. Annealing is seen when complementary sticky ends join.
DNA ligase: enzyme that catalyses the condensation reaction between the phosphate group of one nucleotide and the sugar group of another to form the DNA sugar-phosphate backbone.
Reverse transcriptase: a retroviral enzyme that is able to construct DNA using an mRNA strand as a template .
DNA polymerase: enzyme that synthesises new double stranded DNA – used to make the single stranded cDNA produced by reverse transcriptase double stranded.

3. What is genetic engineering?
Genetic engineering is the term used to describe the processes by which a specific gene is obtained, placed into another organism , causing the recipient to express the new gene.
4 steps to genetic engineering success:
Obtain the required gene
Place a copy of the gene in a vector
Allow the vector to transfer the gene to the target cell
Identify the cells that have successfully been transformed. Allow these to express the new gene though protein synthesis.
Genetic engineering is used for 1 or 2 main reasons: to improve a feature of the recipient organism or to engineer organisms that can synthesis useful products. Inserting a gene into crop plants to give resistance to herbicides will allow a farmer to use herbicides to kill weeds whilst the crop remains unaffected. Inserting a gene for a human hormone such as insulin into bacteria allows large quantities to be produced for human use.

4. There are 3 ways to do this...
Obtaining the gene
There are 3 ways to do this...
Cut the gene out of the genome using a restriction endonuclease enzyme
These enzymes cut through DNA at specific base sequence The sequence is known as the restriction site. Most restriction enzymes catalyse a hydrolysis reaction which breaks the sugar phosphate backbone of the DNA double helix in different places. They give a staggered cut known as a ‘sticky end’ – a short run of unpaired exposed bases.
2. Extract the mRNA of a gene from a cell which makes a lot of that specific protein. Reverse transcriptase enzyme can then be used to convert the mRNA back into a DNA copy of the gene. This is used in the insulin production process covered later in the power point.
3. If the gene sequence is known, the DNA can be made in an automated polynucleotide synthesiser.

5. What are vectors?
A vector is a biological structure able to successfully introduce and integrate a gene into a cell and its genome.
Why do we use vectors?
To make sure that the gene is integrated into the genome and not just deposited into the cytoplasm
Vectors contain genetic control regions to allow the cell to switch on the gene and cause it to be transcribed and translated.
To ensure that the cell is undamaged by the insertion of extra DNA
There are 3 types of vectors that are commonly used in genetic engineering: Bacteria Plasmids, Viruses and Liposomes.
A bacterial plasmid is a double stranded circular DNA molecule that is separate from the main chromosomal DNA.
Plasmids occur naturally in bacteria and carry ‘extra, non essential’ genes that may give a bacteria a survival advantage (like a gene for antibiotic resistance).
Bacteria can exchange plasmids and take up new DNA through a process called conjugation. Bacteria reproduce asexually by binary fission, so usually there is no genetic variation. However, this exchange allows some genetic variation to be introduced, and in times of stress, such as in the presence of antibiotics, the plasmids received may increase chance of survival.
A viral vector ‘infects’ cells by binding to specific complementary receptors on the plasma membrane and then releasing their viral DNA into the cytoplasm.
To use a virus as a vector, the required gene is integrated into the virus and the virus then integrates the gene as it infects the cell.
Non pathogenic viruses are used, but there is always a risk of mutation – the virus could become pathogenic and cause harm.
A liposome is a small sphere of lipid bilayer containing a functioning allele of a gene. The liposome passes through the phospholipid bilayer and delivers the DNA into the cytoplasm of the cell.

6. Putting the gene into the vector: making recombinant DNA
If 2 pieces of DNA have been cut with the same restriction endonuclease enzyme, they will have complementary sticky ends. These unpaired exposed bases can therefore form hydrogen bonds by complementary base pairing. This is called annealing. The enzyme DNA ligase then catalyses condensation reactions which joins the sugar-phosphate backbone of the DNA together to form the recombinant DNA.
Cut with same endonuclease restriction enzyme = Complementary sticky ends
Annealing and DNA Ligase enzyme

7. Transforming the bacteria: getting the recombinant plasmid into the bacteria
Heat shock procedure
Mix the plasmids with the bacteria population
Cool the mixture to near freezing
Add a solution of calcium salts
Quickly raise the temperature to 40°
Usually, only about 1% o the bacteria will take up the plasmid that contains the desired gene.

8. Identifying the transformed bacteria
It is important to notice that not all the plasmids formed in this process will contain the desired gene. We cut all of the DNA with the same restriction endonuclease enzyme, so some plasmids will just reseal to form the original plasmid once again. Three different types of bacteria can therefore be formed. Some bacteria will have taken up no plasmid at all; some bacteria will have taken up a plasmid that does not contain the desired gene and some bacteria (the ones that we want!) will have taken up the recombinant plasmid – the transformed bacteria. We need a way of identifying which are the bacteria with the recombinant plasmid.
The answer is to use plasmid vectors with genetic markers. The original plasmids are chosen because they contain genes that make bacteria resistant to 2 antibiotics – ampicillin and tetracycline. The restriction enzyme that is used to cut the plasmids has its restriction site in the middle of the tetracycline resistance gene. This means that if the required gene is taken up, then the tetracycline resistance gene will be broken up and will not function, but the ampicillin resistance gene will still work.
This plasmid has just resealed to reform the original. The bacteria containing it will be resistant to both ampicillin and tetracycline.
This plasmid has taken up the required gene, so the tetracycline gene is disrupted and the bacteria containing it will not be resistant to tetracycline.

9. Identifying the transformed bacteria:
Replica Plating
1. All of the bacteria are grown on nutrient agar. All of the cells grow to form colonies.
3. A sterile cloth that bacteria can stick to is used to transfer some of the cells from these colonies to agar containing the tetracycline antibiotic.
4. The bacteria that are present on the agar that contains ampicillin must have a plasmid – all those that have died are the bacteria that did not take up a plasmid at all. The bacteria that survived on the tetracycline plate have the original plasmid with both antibiotic resistance genes in tact and therefore these bacteria do not contain the desired gene.
2. A sterile cloth that bacteria can stick to is used to transfer some of the cells from these colonies to agar containing the ampicillin antibiotic.
We therefore want the bacteria that were alive on the ampicillin plate but dead on the tetracycline plate. We can identify these by comparing the plates and the harvest the bacteria containing the recombinant plasmid to produce the product on a large scale.

10. Case study 1: Human Insulin
People who cannot produce enough insulin suffer from type 1 diabetes. This is an autoimmune condition in which the pancreatic Β-islet cells are destroyed. Before genetic engineering was developed, diabetics were treated using pig insulin. There were some problems with this:
Human insulin and pig insulin are not identical, so pig insulin isn’t as effective as human insulin.
Pig insulin can cause adverse reactions in patients
Pig insulin is very expensive to produce
Shortage of insulin because only a very small amount of insulin is present in the pig’s pancreatic tissue
Ethical/moral objections to using animals
Genetic engineering now provides a solution – insulin can be mass produced using bacteria!
Trying to find the one insulin gene in the human genome would be too difficult, so instead we use reverse genetics to get the gene we want.
Separate the cell’s mRNA from all other molecules using basic centrifugation
Use specialised centrifugation to isolate the mRNAs which correspond to the insulin gene by length.
We take mRNA that is present in large concentrations in the B-Islet cells in the pancreas. We work backwards from this, using the enzyme reverse transcriptase, to go from single stranded mRNA to double stranded DNA.

11. Making Human Insulin
Carry out reverse transcription: take the mRNA of the insulin gene and mix with reverse transcriptase enzyme. Add DNA polymerase and free DNA nucleotides to create cDNA.
To allow the insulin gene to join with the cut plasmid, they must have complementary sticky ends. This can be achieved by using the same restriction enzyme to cut the cDNA and the plasmid or by adding artificial sticky ends to those of the plasmid.
3. The vector is prepared by cutting the plasmid with the same restriction enzyme as was used to cut the cDNA.

12. Making Human Insulin Plasmid Insulin DNA
4. The insulin DNA and plasmid are mixed together with Ligase enzyme. The complementary sticky ends anneal and the Ligase enzyme joins the sugar-phosphate backbone. A recombinant plasmid is formed.
Insulin DNA
Plasmid
5. Heat shock – recombinant plasmid introduced into bacteria
6. Identify the transformed bacteria using replica plating
7. The transformed bacteria containing the insulin gene can produce insulin for human use!

13. Case Study 2: Golden Rice
Vitamin A deficiency can cause serious health problems, including Xerophthalmia . Vitamin A is needed for:
Maintaining vision at low light intensities
Effective immune system
Large numbers of people in areas such as Africa and India, where the staple food is rice, suffer from the effects of vitamin A deficiency.
Genetic engineers therefore found a way to add genes to the rice plant that would allow for the transcription and translation of the beta-carotene in the endosperm that is eaten by humans.
Vitamin A itself comes only from animal sources, but it can be synthesised in the gut from beta carotene – a precursor. Beta carotene is a photosynthetic pigment, so it is only expressed in the parts of the rice plant that photosynthesise – this is the hull and bran layers. We only eat the endosperm, in which the beta carotene genes are switched off.

14. Case Study 2: Golden Rice The genes codes for the following enzymes:
Most of the enzymes needed for the synthesis of beta carotene were already present in the endosperm. The insertion of 2 genes into the rice genome was needed in order for the metabolic pathway to be activated in the cells of the endosperm.
Precursor molecules
Phytoene
Lycoprene
Beta Carotene
The genes codes for the following enzymes:
From daffodils
Phytoene Synthetase
Crt1 enzyme
From bacteria
Already present in endosperm
Other Enzymes
The first version of golden rice contained 4 x more beta carotene than normal rice, but this still meant that you’d have to eat large amounts of rice every day in order to prevent vitamin A deficiency.
Further genetic engineering added stronger promoters to the vitamin A genes. These promoters increased the transcription of the beta-carotene synthesising enzymes, so ‘Golden Rice 2’ contained over 20x the original amount of beta carotene in the endosperm.