Transcript Biotechnology – Biotechnological techniques

Biotechnology –
Biotechnological techniques
1. Use of micro-organisms
2. Industrial production of enzymes
3. Tissue cultures
Use of micro-organisms
 Growing micro-organisms
 Stages of growth
 Diauxic growth
Growing Micro-organisms
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
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
Growth conditions
 The following need to be considered
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Growth media
Temperature
pH
Oxygen
 Obligate aerobes / anaerobes?
 Facultative
 Carbon dioxide
Growth conditions cont…
 Large scale growth of microorganisms can occur in a fermenter
 Growth conditions are controlled in
the fermenter
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.
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
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)
Stages of Growth
Stages of Growth
 Typically microbial growth falls into four
stages
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
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
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
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
Measuring Cell growth cont..
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)
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)
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
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
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.
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.
• Microbial growth occurs exponentially
(logarithmically). In other words, with
every generation the number of cells
double:
21
(Generation 1)
2 cells
22
23
(Generation 2)
(Generation 3)
4 cells
8 cells
24
(2n)
(Generation 4)
16 cells
n = the number of generations
•
The time taken for a population to double is called
the generation time (g)
• 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
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.
• 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
Diauxic Growth
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.
 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
Diauxic Growth
I = growth on
glucose
II = growth on
lactose
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.
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
Diauxic Growth
 What happens when E. Coli is using
lactose as a source of energy?
 Remember
Lactose β-galactosidase galactose + glucose
 Use the term inducer in addition to the
previous terms
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
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
Diauxic Growth
 The presence of glucose inhibits the
production of cAMP
Diauxic Growth
 When glucose is absent, cAMP levels rise.
 cAMP binds to CAP (activator protein)
 CAP then binds upstream from operator and
transcription occurs
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)