+ 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: 1. The required gene is obtained. 2. A copy of the gene is placed into a vector. 3. The vector carries the gene to the recipient cell. 4. 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.

KEY: Gene from Human Gene from E.coli This gene (from a human) can be cut with a restriction enzyme such as EcoRI Sticky End If this section of DNA from E.coli is also cut with EcoRI, a complimentary sticky end is produced. This is a section of DNA from E.coli. 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 1. Explain why different restriction enzymes have different restriction sites (recognition sequences). 2. Explain why restriction enzymes can be a useful defense mechanism for bacteria against viruses. 3. If bacterial DNA contains base sequences that are the same as the restriction sites of their enzymes, these sites are methylated (-CH 3 group added). Explain why. 4. 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.vector DNA coding for a desired protein Restriction Endonuclease A plasmid Restriction Endonuclease 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. This is ‘Step 2’ (insertion) in the process of making a protein using gene technology

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. Calcium ion medium 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. 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

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. Gene for resistance to ampicillin Gene for resistance to tetracycline 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? 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. This agar however, has been treated with tetracycline Colonies are allowed to develop ? 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!

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

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.