F215: Biotechnology And Gene Technologies Flashcards

Question

Describe the standard growth curve for a culture of microorganisms.

Answer 1

Page 160 Lag - constant population, adjust to conditions surrounding them. Reproduction = death Log - population increases as space and nutrients increase. Reproduction > death Stationary - nutrients decrease, waste products increase, death rate = reproduction rate. If an open system his is the carrying capacity. Decline - nutrients are exhausted, levels of toxic waste increase, death rate > reproduction rate. All organisms will die in a close system eventually.

Answer 2

The sum total of all the chemical reactions that go on in an organism.

Answer 3

Substances produced by an organism as part of it's normal growth, like amino acids, proteins, enzymes etc. The production of primary metabolites matches the growth in population of the organism.

Answer 4

Substances produced that are not part of it's normal growth, like anti biotic chemicals. Production occurs after the main growth period of the organisms and does not match the growth in population of the organism. Only a small number of microorganisms produce secondary metabolites.

Answer 5

Temperature - enzymes Type and time of addition of nutrient O2 conc - do not want anaerobic respiration to occur usually. pH - reduce activity of enzymes.

Answer 6

Batch: Starter population of a micro organism is mixed with a specific quantity of nutrient solution, they are allowed to grow with no further nutrients added. Eg penicillium fungus Continuous: Nutrients are added and products are removed at regular intervals, or continuously. Eg insulin E. coli bacteria

Answer 7

The absence of unwanted microorganisms.

Answer 8

Any measure taken at any point in a biotechnological process to ensure that unwanted microorganisms do not contaminate the culture that is being grown or the products that are extracted.

Answer 9

Batch: Growth rate is slower because nutrient level declines with time. Easy to set up/maintain/control conditions. If contamination occurs only one batch is lost. Less efficient, the fermenter is not in operation all of the time, cleaning etc. Very useful for processes involving secondary metabolites being produced.

Answer 10

Growth rate is higher as nutrients are continuously added to the fermentation tank. Set up is difficulter, maintenance of required growing conditions is harder to achieve. If contamination occurs huge volumes of product can be lost. More efficient as it operates all the time. Very useful for processes involving primary metabolite production.

Answer 11

A contaminant.

Answer 12

They compete with the culture microorganisms for nutrients and space. Thus they Reduce the yield of useful products from the culture microorganisms. May cause spoilage of the product. May change the conditions, eg by a product changing the pH. May produce toxic chemicals. May destroy the culture microorganism and their products.

Answer 13

Lab level: Sterilising apparatus either in a flame or by UV light. Carrying out work in a fine cuboard, so air circulation carries away harmful airborne contaminants. Cultures of microorganisms kept closed where possible. Large scale: Washing/disinfecting fermenters when not in use to remove excess nutrients and kill microorganisms. Make fermenters of stainless steel to prevent microbes sticking to it. Sterilising all nutrients before adding to the fermenter prevents adding contaminants.

Answer 14

Any technique where enzyme molecules are held, seperate a from the reaction mixture. Substrate molecules can bind to the enzyme molecules and the products formed go back into the reaction mixture leaving the enzyme molecules in place.

Answer 15

Specificity - only react with specific chemicals in a huge mixture, fewer by products are formed. Temperature - most function at low temperatures, saving money on fuel. Not used up No waste products created

Answer 16

The extraction of enzymes from the fermentation mixture. | ????

Answer 17

Advantages: Downstream processing costs are low. Enzymes are immediately available for further use, good for continuous. More stable than regular enzymes as the immobilising matrix protects the enzyme. Disadvantages: Immobilisation requires time, materials etc. Is more expensive. Less active than regular enzymes as they do not mix freely with the substrate. Less complexes formed. Contamination is costly as the whole system needs to be stopped??

Answer 18

Enzymes are mixed with the immobilising support and bind to it due to hydrophobic interactions and ionic links. The bonds are not that strong so enzymes can become detached (leakage). Can give high reaction rates.

Answer 19

Adsorption Covalent bonding Entrapment Membrane seperation

Answer 20

Enzymes are covalently bonded to a support using a cross linking agent. Does not immobilise a large quantity of enzyme but the binding is very strong so there is little leakage. Page 165

Answer 21

Enzymes are trapped in a gel brad or network of cellulose fibres. Trapped in the natural state so active site is not affected. Reaction rate decreases because substrate molecules need to get through the trapping barrier. Active site is thus less available than with adsorbed/covalently bonded enzymes.

Answer 22

Enzymes are physically separated from the substrate mixture by a partially permeable membrane. The enzyme solution is held at one side of a membrane whilst the substrates pluton passes along the other side ???

Answer 23

The study of the whole set of genetic information in the form of the DNA base sequences that occur in the cells of organisms of a particular species. The sequences genomes of organisms are place on public access databases.

Answer 24

Coding DNA, and mostly non-coding DNA.

Answer 25

They have regulatory functions, many of which we are still discovering.

Answer 26

Genomes are mapped to identify which chromosome or section of chromosome they have come from. Samples of the genome are sheared (broken) into smaller sections. "Shotgun" These sections are placed into separate bacterial artifices chromosomes (BACs) and transferre to E. coli cells. As the cells grow in culture, cloned are produced - these are clone libraries. The BAC section is then sequenced. (Another card)

Answer 27

Cells containing BACs are cultured. The DNA is extracted from the cells and the restriction enzymes use to cut it into smaller fragments. By using different restriction enzymes different fragment types are given. The fragments are separated by electrophoresis. Each fragment is sequenced. Computer programs then compare overlapping regions from the cuts made by different restrictions enzymes in order to reassemble the whole BAC segment sequence.

Answer 28

The identification of genes for proteins found in all or many living organisms gives clues to the importance of certain genes for life. Comparing DNA can show evolutionary relationships. Modelling the effects of changes to DNA. Developing effective vaccines/drugs by looking at pathogenic and similar non pathogenic to identify the genes that cause the disease. Individuals DNA can be studied to identify mutant alleles, and identity particular diseases that may be common for this.

Answer 29

Used to separate DNA fragments based on their size. | Used to separate for identification and analysis.

Answer 30

Uses a gel plate containing agarose (sugar) which is covered in buffer solution. Electrodes are at each end so a current is passed through it. Longer strands get caught in the gel and are slowed, where short strands move quickly through the gel. DNA samples are split into fragments using restriction enzymes. The DNA samples are place into wells cut into the negative end of the gel. The gel is immersed in a tank of buffer solution and an electric current is passed through for a fixed period of time. DNA is negatively charged due to the phosphate groups, and is attracted to the positive end. Shorter lengths move faster so move further. The position of fragments can be shown using a dye that stains DNA molecules. Page 168

Answer 31

A nylon/nitrocellulose sheet is placed over the gel, covered in paper towels, pressed and left overnight. The DNA fragments are transferred to the sheet and can now be analysed.

Answer 32

A short single stranded piece of DNA that is complementary to a section of DNA being investigated. It can be labelled in two ways: Using a radioactive marker so it's location is revealed by exposure to photographic film. Using a fluorescent marker that emits colour when exposed to UV light.

Answer 33

Copies of the probe can be added to any sample of DNA fragment, and because they are single stranded, they will bind to any fragment with a complementary base sequence. The complementary base pairing is annealing.

Answer 34

Locating a desired gene wanted for genetic engineering. Identifying the same gene or a variety of genes when comparing genomes. Identifying the presence I absence of an allele for a particular genetic disorder.

Answer 35

Short single stranded sequences of DNA. They are needed in sequencing reactions and polymerase chain reactions to bind to a section of DNA, because the DNA polymerase enzymes cannot bind directly to single stranded DNA fragments. They are added to start the process.

Answer 36

Polymerase chain reaction. "Artificial DNA replication" Makes many copies of the sample.

Answer 37

The fact that DNA: Is made from antiparallel backbone strands. Made of strands that have a 5' end and a 3' end. Grows only from the 3' end. Base pairs pair up according to complementary base pairing.

Answer 38

Forensics, to generate enough material for genetic profiling.

Answer 39

Can only replicate short sequences of DNA, not entire chromosomes. A cycle of heating and cooling is used to desperate and bind strands, DNA helipads separates strands in the natural process.

Answer 40

The DNA sample is mixed with a supply of DNA nucleotides and DNA polymerase. The mixture is heated to 95*c, breaking hydrogen bonds that were keeping the complementary strands together. Now they are single stranded. Short lengths of single stranded DNA are added (primers). The temperature is reduced to 55*c, Allowing the primers to form hydrogen bonds and form small sections of double stranded DNA at either end of the sample. DNA polymerase can bind to these double stranded sections. Temperature is raised to 72*c (optimum temp for DNA polymerase). The enzyme extends the double stranded section by adding free floating nucleotides to the unwound DNA. When the DNA polymerase reaches the other end of the DNA strand, a new double stranded DNA molecule is generated. The amount of DNA increases exponentially.

Answer 41

DNA polymerase is not denatured by extreme temperatures used in he process. It is derive from a thermophilic bacterium found in hot springs around 90*c.

Answer 42

The primer anneals at the 3' end of the template strand, allowing DNA polymerase to attach. DNA polymerase adds free nucleotides according to complementary base pairing so the strand grows. When a modified nucleotide is added the polymerase enzyme cannot continue and that reaction stops. This happens on many different template strands, each ending with known nucleotides that are fluorescently marked. The strands run through a computer and a laser reads where the strands end by identifying the marker, it then knows one base since It is complementary. It does this for the entire sequence. Page 173

Answer 43

A section of DNA, often in the form of a plasmid, which is formed by joining DNA sections from two different sources.

Answer 44

Obtaining a specific gene, Placing that gene in another organism through a vector, The recipient then expresses that gene through protein synthesis

Answer 45

It is formed when DNA is cut using a restriction enzyme. It is a short run of unpaired, exposed bases seen at the end of the cut section. Complementary sticky ends can anneal (bases pair together) as part of the process of recombining DNA fragments.

Answer 46

Used to cut DNA where've a specific base sequence occurs and only where that sequence occurs. This is the restriction site. The enzyme catalyses a hydrolysis reaction which breaks the phosphate sugar backbones of the DNA double helix in different places. This exposes some bases - sticky ends.

Answer 47

Catalyses a condensation reaction which joins the phosphate sugar backbones of the DNA double helix together. Both fragments must have been cut with the same restriction enzyme, so that both sticky ends are complementary, and the bases can pair up.

Answer 48

An organism that contains DNA that ha been added to it's cells as a result of genetic engineering.

Answer 49

Improving a feature of the recipient organism - Eg inserting a gene into a crop to give resistance to herbicides. Or a gene that improves muscle growth into livestock. Engineering organisms that can synthesise useful products - Eg insulin hormone into bacteria to produce insulin for humans. Golden rice and shit.

Answer 50

Bacterial plasmids.

Answer 51

A circular piece of DNA. | Separate from the main bacterial chromosome.

Answer 52

In the presence of ligase many will reseal to reform the original plasmid.

Answer 53

Calcium salts are added, and the temperature is lowered to freezing, then quickly raised to 40*c. This increases the rate at which plasmids are taken up by the bacterial cells, but the process is still very inefficient. Those that do are transformed bacteria.

Answer 54

Bacterial cells can join together and pass plasmid DNA from one bacterial cell to another. This process can take place between bacteria of different species and is concerning since bacteria often carry genes that are resistant to antibiotics. It thus speeds up the spread of antibiotic resistance between bacterial populations.

Answer 55

It contributes to genetic variation and survival In the case of antibiotic resistance.

Answer 56

Some bacteria do not take up the plasmid. Some Bacteria that have taken up the plasmid but have not sealed in a copy of the gene but has sealed up on itself to reform the original plasmid. Some bacteria take up the recombinant plasmid - these are transformed bacteria. No visual difference.

Answer 57

The mRNA for insulin is located, and reverse transcriptase is used to synthesis a complementary DNA strand. Adding DNA polymerase and a supply of DNA nucleotides to the single strands means the second strand is built on using the copied DNA as a template. This produces a copy - cDNA. Unpaired nucleotides are added to give sticky ends complementary to those on the cut plasmid. Plasmids from E. coli (from human intestine) are then cut open with a restriction enzyme and mixed with the cDNA genes. DNA ligase then seals up the plasmids which take up the gene - now recombinant. The plasmids are mixed with bacteria, some of which take up the recombinant plasmids. The bacteria are grown on an agar plate, where each bacterial cell grows to produce a mound of identical cloned cells (colony).

Answer 58

The original plasmids carry genes that make them resistant to two antibiotic chemicals (ampicillin and tetracycline). These resistance genes are genetic markers, since the E. coli is susceptible to both the antibiotic chemicals. The plasmids are cut by a restriction enzyme that has it's target sit in the middle of the tetracycline resistance gene, so this gene will no longer work if the insulin gene is taken in.

Answer 59

The process of growing bacteria on an agar plate, then transferring a replica of that growth to other plates, usually containing different growth promoters or inhibitors. Analysis of growth patterns on the replica plates gives information about the genetic properties of the growing bacteria.

Answer 60

The bacteria are grown on standard nutrient agar, so bacterial cells grow to form colonies. Some cells from the colonies are transferred into agar made with ampicillin, so only those than have taken up the plasmid grow. Some cells are then transferred onto agar that has been made with tetracycline so only those that have taken up a plasmid without an insulin gene will grow. (Only non recombinant) By keeping track of which colonies are which, we can look at which colonies grew on ampicillin but not on tetracycline. Since these are the recombinant colonies. These are then grown on a large scale.

Answer 61

Insulin mRNA is only produced in those cells which transcribe the insulin gene, these are the beta cells in the islets of Langerhans in the pancreas.

Answer 62

Something that contains a higher than normal concentration of a particular nutrient. Such as Golden Rice and beta carotene.

Answer 63

The only source is through meat. Required for the formation of rhodopsin. Involved in the synthesis of glycoproteins. Needed for the maintenance and differentiation of epithelial tissues and help to reduce infection. Essential for bone growth. Lack of vitamin A gives a deficiency disorder. Leads to blindness. Made from beta carotene in the human gut. Mainly in less developed countries.

Answer 64

Beta carotene is a precursor, which is converted to active vitamin A in the gut. Vitamin A is also fat soluble, so lipids must be absorbed in order for the vitamin to be taken up.

Answer 65

The part that is eaten (endosperm/grain) has the gene for beta carotene switched off.

Answer 66

It was found 2 genes need to be inserted for the metabolic pathway to be activated in the endosperm cells to synthesise beta carotene: Phytoene synthetase - extracted from daffodil plants. Crt1 enzyme coding for carotene desaturase (extracted from the soil bacterium Erwinia uredovora) These genes are inserted into plasmids with promoters. The plasmids were inserted into bacteria called Agribacterium tumefaciens. Mix the bacteria with rice embryos and they will infect some embryos so they carry the beta carotene gene. The rice embryos now containing the beta carotene genes will grow into adult plants which will produce seeds containing beta carotene in their endosperm.

Answer 67

Precursor molecules + Phytoene synthetase --> Phytoene Phytoene + crt1 enzyme ---> lycopene Lycopene + enzymes ---> beta carotene Page 181.

Answer 68

The amount of rice in which you would need to eat is huge. Lipids must also be taken up in order for the vitamin A to be absorbed properly, in developing countries this is not easily come by.

Answer 69

Reduction in biodiversity. Safety is yet unknown. Damage environment? GM rice could breed with wild types and contaminate wild rice populations.