Biotechnology – Biotechnological techniques

1. Biotechnology – Biotechnological techniques Use of micro-organisms Industrial production of enzymes Tissue cultures

2. Use of micro-organisms • Growing micro-organisms • Stages of growth • Diauxic growth

3. Growing Micro-organisms

4. Obtaining pure cultures • A pure culture comes from a single organism or colony • There are three methods of creating single colonies • Single colony isolate • The plate is streaked to ‘thin’ out bacteria producing single colonies at the end of the streak

6. Obtaining pure cultures cont… • Spread plate technique • The culture is aseptically diluted • The diluent is placed on the surface of the agar and spread using a spreader • Pour plate technique • Cells are diluted • They are added to a petri dish • Molten agar (at 45oC) is added to petri dish and allowed to set

7. Growth conditions • The following need to be considered • Growth media • Temperature • pH • Oxygen • Obligate aerobes / anaerobes? • Facultative • Carbon dioxide

8. Growth conditions cont… • Large scale growth of micro-organisms can occur in a fermenter • Growth conditions are controlled in the fermenter

10. Scaling up • Obtain a pure culture • Transfer a sample from a single colony to growth medium • Investigate suitable conditions for growth • Monitor growth in different conditions (see measuring cell growth) • Once optimum conditions are determined the culture needs to be maintained and subcultured.

11. Scaling up cont… • Volume of culture is scaled up from a petri dish to a flask to a fermenter (often starting at pilot plant size and then to industrial size fermenters) • The culture grown at one stage will form approximately 1-5% of the volume at the next stage of the scale up process

12. Scaling up cont… • Conditions are monitored using probes • Conditions are regulated to maintain optimum growth • Purity of the culture is checked at each scale up. • See later for other points on industrial production (growth and products)

13. Stages of Growth

14. Stages of Growth • Typically microbial growth falls into four stages

15. Stages of growth cont… • Lag phase • Initial inoculation into liquid culture • Period of adaptation occurs to build up levels of metabolites and repair damage • The length of time varies depending on the source of the culture and its previous growing conditions

16. Stages of growth cont… • Exponential phase • Reproduction by binary fission (1 cell divides into 2 cells) • Population doubles with each successive generation • Exponential increase is observed

17. Stages of Growth cont… • Stationary phase • Growth slows due to nutrients becoming exhausted and build up of toxic metabolites • Death phase • Nutrients become completely exhausted and cells use up internal energy stores

18. Measuring cell growth • Cell counting • Direct count using a microscope • A haemocytometer can aid counting • A fixed volume of culture is added to the slide • The number of cells per ml can be calculated

19. Measuring Cell growth cont..

20. Measuring cell growth cont… • Advantages • Quick • Disadvantages • Dead and live cells counted (total count) • Small cells can be missed • Unreliable at low cell densities (few bacteria will be seen if the concentration is lower than 106 cells per ml)

21. Measuring cell growth cont… • Dilution plating • Bacteria are diluted by a known factor (serial dilution if more than 1 step is involved) • A known volume of the diluted culture is plated (see pour plate technique / spread plate technique) • Number of organisms can be calculated by average number of colonies per plate ÷ dilution factor x volume plated (in ml) • Gives a count of the living organisms (a viable count)

22. Measuring cell growth cont… • Advantages • Best information on viable cells • High sensitivity • Disadvantages • Small errors are amplified by the dilution process • Delay for results (1-5 days depending on the organism) • Most accurate if there are 30 – 300 colonies on a plate. More or less may require the test to be repeated

23. Measuring cell growth cont… • Turbidity • Bacteria in a liquid culture make the liquid turbid • The more bacteria the greater the turbidity • A colorimeter can be used to measure absorbance / transmission through the sample • Gives an indirect count

24. Measuring cell growth cont… • Advantages • Quick, easy and does not destroy the sample • Disadvantages • Non-viable cells also contribute to turbidity • Calibration needs to be carried out for each type of bacteria. A standard curve is created for each organism relating a direct count to absorbance.

25. The Importance of Measuring Cell Growth • Knowledge of microbial growth rates are important to research and to industry. They enable microbiologists to control cell growth and so allow scientists to study cell behaviours and to produce maximum quantities of commercial products e.g. antibiotics.

26. Microbial growth occurs exponentially (logarithmically). In other words, with every generation the number of cells double: 21 22 23 24 (2n) (Generation 1) (Generation 2) (Generation 3) (Generation 4) 2 cells 4 cells 8 cells 16 cells n = the number of generations • The time taken for a population to double is called the generation time (g)

27. Generation time (g) can be worked out if the number of generations (n) and the time (t), usually in hours, is known. g=t/n • For example: what is the generation time if, 20 generations have occurred in 6 hours. g=6/20 g=0.3 hours

28. Calculation of growth rate constant It is also useful to be able to calculate the growth rate constant (k) • Growth rate constant, k, is a measure of the number of generations (the number of doublings) that occur per unit of time in an exponentially growing culture.

29. The formula for this is k = ln2 / g • where ln 2 is the natural log of 2 (determine this from your calculator) • Note ln2 = 0.693 For example: A microbial culture that took 10 mins (0.17h) to double (g), would have a growth rate constant: K = 0.693/0.17 = 4.1h-1

30. Diauxic Growth

31. Diauxic Growth • Diauxic Growth is a form of growth that occurs when there are 2 carbon sources for metabolism. • Although both carbon sources are available for bacteria, they will have a preference for one type of carbon source, usually glucose. Only once it has been depleted do the bacteria utilise the other carbon source e.g. Lactose.

32. This process is brought about by catabolite repression – the presence of glucose suppresses the synthesis of enzymes needed to metabolise lactose. • It results in a two-step growth curve

33. Diauxic Growth I = growth on glucose II = growth on lactose

34. Diauxic Growth • Why do cells use catabolite repression? Glucose can be metabolised more quickly than other carbon sources and so if bacteria use catabolite repression they can reproduce at their maximum rate in any environment.

35. The lac operon example from Higher......... • What happens when E. Coli is using glucose as a source of energy? • Use the terms repressor molecule, regulator gene, operator and structural gene

36. Diauxic Growth • What happens when E. Coli is using lactose as a source of energy? • Remember Lactose galactose + glucose • Use the term inducer in addition to the previous terms β-galactosidase

37. Diauxic Growth • What happens when both glucose and lactose are available as a source of energy? • E. Coli grows on glucose only • The repressor molecule still binds to lactose and not the operator but… there is another level of control

38. Diauxic Growth • For the β-galactosidase gene to be transcribed an activator protein needs to bind upstream from the operator • This activator protein is known as CAP • CAP is present in an inactive form in the bacteria cell • It is activated by the binding of cAMP

39. Diauxic Growth • The presence of glucose inhibits the production of cAMP

40. Diauxic Growth • When glucose is absent, cAMP levels rise. • cAMP binds to CAP (activator protein) • CAP then binds upstream from operator and transcription occurs

41. Lac Operon control - summary There are 2 control elements that are needed to give expression of the gene: • Removal of Negative control • Repressor molecule does not bind to the operator in the presence of lactose • Positive control • Binding of CAP/cAMP complex (which acts as a positive effector)