module 2 – biotechnology & gene technologies genetic engineering module 2 – biotechnology & gene technologies

learning objectives Understand what is meant by genetic engineering. Understand the steps involved. Understand what restriction enzymes do. Understand why sticky ends are important.

success criteria State the definition of genetic engineering. Describe what restriction enzymes do. Explain the importance of sticky ends.

From the spec

starter

starter 2 Try to come up with a definition for the term – genetic engineering. The definition: The transfer of genes from one organism to another (often a different species). The organism receiving the gene expresses the gene product through protein synthesis.

genetic engineering Genetic engineering is a rapidly advancing field of Biology. We can now manipulate, alter and even transfer genes from one organism to another. The ability to do these things has proved invaluable in the industrial and medical sectors.

requires... The following steps are necessary: The required gene is obtained. A copy of the gene is placed into a vector. The vector carries the gene to the recipient cell. The recipient expresses the gene through protein synthesis.

sticky ends

cutting the genes out In order to isolate a gene, it needs to be cut from the donor organisms DNA. This is done using ‘molecular scissors’ known as restriction enzymes. Cuts made with restriction enzymes can have two results: Some restriction endonuclease produce ‘blunt ends’ Some restriction endonuclease produce ‘sticky ends’

importance of sticky ends Restriction enzymes that cut the sugar-phosphate backbone in different places, produce sticky ends. These are really important due to the exposed bases left at the staggered cut. Due to the complimentary nature of DNA bases, sticky ends on one gene, will pair up with sticky ends on another bit of DNA, - provided it has also been cut with the same restriction enzyme.

This is a section of DNA from E.coli. This gene (from a human) can be cut with a restriction enzyme such as EcoRI KEY: Gene from Human Gene from E.coli Sticky End This is a section of DNA from E.coli. If this section of DNA from E.coli is also cut with EcoRI, a complimentary sticky end is produced. Sticky End If these two ‘cut’ pieces of DNA are mixed, recombinant DNA has been produced.

Once the bases have paired, they form their usual weak hydrogen bonds between each other. The only thing left to do, is form the link between the sugar-phosphate backbones, and this is done by the enzyme, DNA Ligase.

Questions Explain why different restriction enzymes have different restriction sites (recognition sequences). Explain why restriction enzymes can be a useful defense mechanism for bacteria against viruses. If bacterial DNA contains base sequences that are the same as the restriction sites of their enzymes, these sites are methylated (-CH3 group added). Explain why. The restriction enzyme EcoR1 was the first restriction enzyme isolated from E. coli. Suggest how restriction enzymes are named.

plenary

Exam practice

Answers

learning objectives Understand what is meant by genetic engineering. Understand the steps involved. Understand what restriction enzymes do. Understand why sticky ends are important.

success criteria State the definition of genetic engineering. Describe what restriction enzymes do. Explain the importance of sticky ends.

Insertion of DNA into a vector VECTOR – used to transport DNA into a host cell. PLASMID – the most commonly used vector. A circular piece of DNA found in bacteria. Plasmids are useful because the nearly always contain antibiotic resistance genes (see later).

The Plasmid One of the antibiotic resistant genes is disrupted when the restriction enzymes cuts open the plasmid. The other antibiotic resistant gene is used in selection of the correct host cells. (See later)

Insertion into plasmids What combinations of plasmid will form?

Inserting genes into Plasmids The real-life application of what we have just learnt, occurs when geneticists insert an animal or plant gene into plasmids. Plasmids are small loops of DNA which are found in addition to the large circular chromosome that bacterial cells possess. By inserting our chosen gene into a plasmid, the plasmid acts as a ‘carrier’, or vector, which we can then introduce back into a bacterial cell. This is ‘Step 2’ (insertion) in the process of making a protein using gene technology Restriction Endonuclease Restriction Endonuclease REFER TO HANDOUT 2 A plasmid DNA coding for a desired protein As the DNA fragment was cut out using the same restriction endonuclease as used to cut the plasmid open, they have complimentary sticky ends. Remember, that DNA Ligase would once again be used to bond the sugar-phosphate backbones.

Discussion questions Why was it important to find an enzyme that would cut once in the plasmid? What other considerations were there in choosing the enzyme to cut the plasmid and DNA sequence (think shaded areas). How can we use the new recombinant plasmid to produce insulin?

Introducing our recombinant plasmids into host cells Introducing recombinant plasmids into bacterial cells is called transformation. This is done by mixing the plasmids with the cells in a medium containing calcium ions, and changing the temperature The calcium ions make the bacterial cells permeable, allowing the plasmids to pass through, into the cell. However, only a few bacterial cells (approx 1%) will actually take up the plasmids. For this reason, we need to identify which ones have been successful. This is done with gene markers. Calcium ion medium This is ‘Step 3’ (transformation) of producing a protein by DNA technology

Bacterial chromosome plasmid Insulin

Using Gene Markers to identify successful host cells... There are a number of different ways of using gene markers to identify whether a gene has been taken up by bacterial cells. They all involve using a second, separate gene on the plasmid. This second gene acts as a ‘marker’ because.... It may give resistance to an antibiotic It may make a fluorescent protein that is easily seen It may produce an enzyme whose action can be identified Each of the 3 above mechanisms are actual methods of employing gene markers to identify bacterial cells that have taken up plasmids... They will be discussed on the next slides...

1. Antibiotic-Resistance Markers Many bacteria contain antibiotic resistance genes in their plasmids. Some in fact, can have two genes for resistance to two different antibiotics, in the same plasmid. Any bacterial cell possessing this plasmid, would be resistant to both of the antibiotics, ampicillin and tetracycline. But what if we cut right in the middle of the tetracycline-resistance gene (with a restriction endonuclease), and insert a gene of our own interest? Gene for resistance to tetracycline Gene for resistance to ampicillin Bacteria with this plasmid would only be resistant to ampicillin, not tetracycline. How is this of any advantage to us?

First, the recombinant plasmids are introduced into bacterial host cells (transformation)

The bacteria are grown on agar plates treated with ampicillin Colonies are allowed to grow, but will only do so if they are resistant to ampicillin – i.e. Bacteria that took up the plasmid. A replica plate is now made. This is when you literally press the agar of one Petri-dish, onto the agar of a new Petri-dish, transferring bacterial cells from each colony onto the new agar. There is a missing colony, which has lost resistance to tetracycline. This must be a colony of cells which have taken up the recombinant plasmid! This agar however, has been treated with tetracycline ? Colonies are allowed to develop

2. Fluorescent Markers This is a more recent method of finding out whether bacteria have taken up the desired plasmids. Throughout nature, there are organisms such as jellyfish, that produce fluorescent proteins. These proteins, coded for by their own genes, can be isolated and then introduced into bacterial cells via vectors. The range of natural fluorescent proteins can be seen on this Petri-dish. First the fluorescence gene is inserted into a plasmid vector Using restriction enzymes, the gene of interest (e.g. Human insulin gene) is then inserted into the middle of the fluorescence gene, so the latter can no longer be expressed Bacteria that have taken up the plasmid alone will fluoresce under a microscope BUT those containing the recombinant plasmid will not fluoresce This is an easier way to identify bacteria expressing the gene of interest All you have to do is first insert your gene of interest (such as insulin) into a gene for a fluorescent protein. Then insert this insulin/fluorescence hybrid gene into a plasmid vector. Then transfer the plasmids into bacterial cells! Any cells that successfully took up plasmids, will be glowing on your Petri-dish!

3. Enzyme Markers This method involves inserting your gene of interest (e.g. Insulin), into a gene that codes for an enzyme such as lactase. There is a particular substrate that is usually colourless, but turns blue when lactase acts upon it. If you insert your chosen gene into the gene that makes lactase, you will inactivate the lactase gene. If you now grow bacterial cells on an agar medium containing the colourless substrate, any bacteria that have taken up the recombinant plasmid, will form white colonies not blue ones. Therefore any colourless colonies on the agar plate indicate to you, which cells have been transformed.

Gene Therapy What are some of the ethical considerations for using gene therapy?  --Some Questions to Consider... What is normal and what is a disability or disorder, and who decides? Are disabilities diseases? Do they need to be cured or prevented? Does searching for a cure demean the lives of individuals presently affected by disabilities? Is somatic gene therapy more or less ethical than germline gene therapy? In cases of somatic gene therapy, the procedure may have to be repeated in future generations. Preliminary attempts at gene therapy are exorbitantly expensive. Who will have access to these therapies? Who will pay for their use?

Cystic fibrosis is the most common inherited disease in the UK. It affects 1 in every 2500 people.

1 in 25 people carry the faulty gene that causes cystic fibrosis.

What is gene therapy? Gene therapy replaces defective genes with healthy ones to treat genetically inherited diseases. The cause of the disease is tackled not just the symptoms. Cystic fibrosis is an inherited disease that can be treated by gene therapy. Gene therapy has been used in the treatment of cancer and recently, in 2008 to treat genetic blindness

Cystic fibrosis Cystic fibrosis is a recessive genetic disease which affects 1 in 2500 people in the UK. 1 in 20 are carriers! Symptoms include: Production of thick sticky mucus from the cells that line respiratory system causes congestion of the lungs, breathing difficulties and higher risk of infection. Blockage of the pancreatic duct with mucus can lead to a reduced release of pancreatic juice and inadequate digestion. prevents pancreatic enzymes from reaching the duodenum and leading to the formation of fibrous cysts. The reproductive system is also blocked, preventing sufferers having children.

Treatment using gene therapy There are 2 ways in which gene therapy can be used to treat cystic fibrosis Gene replacement – defective genes are replaced by healthy ones Gene supplementation – one or more copies of the healthy gene are added alongside the defective gene. The added genes are dominant so they mask the effects of the recessive alleles.

Gene therapy techniques 1) Germ-line therapy – replacing/supplementing the defective gene in the fertilised egg. All cells will develop normally and the cells of the offspring 2)Somatic-cell therapy – targets the affected tissues, such as the lungs, . Long term is to target undifferentiated stem cells so treatment is effective for the life span of the individual. Name at least one issue with each type of therapy Moral and ethical issues Treatment needs to be repeated – limited success

How can somatic-cell gene therapy help? It involves inserting cloned functional CFTR genes into epithelial cells of the lungs to replace defective genes. 2 techniques are being tested: (1) Wrapping CFTR genes in lipids that can be absorbed through the cell surface membranes. (2) Inserting CFTR genes into harmless viruses called adenoviruses that are then allowed to ‘infect’ the cells.