Download Biotechnology

Biotechnology
The use of living organisms (usually
bacteria) to provide us with a substance or
a process.
Uses are generally in industry
(manufacturing or food) or in medicine
• Thiobacillus ferrioxidans is used to oxidise
the metal sulphides into metal sulphates.
• The sulphate is then soluble in water so it
can be washed out of the rocks.
• This is called bioleaching.
• The bacteria can usually survive in highly
acidic conditions and in a range of
temperatures.
Industrial applications of
microorganisms
• Some bacteria are chemoautotrophic (get
energy from the breakdown of inorganic
chemicals).
• Metals are found in the ground as metal
sulphides (heavy metals)
• Bacteria are used to change these in to pure
metals
• Metals which can be extracted in this way
– copper, uranium, cobalt, lead, nickel and gold.
• Bacteria of the genus Thiobacillus are used
• Advantages of using bacteria to mine ores
in this way:
• – Getting metal from low grade ore in
sufficient quantities to make a profit
• – Extract valuable wastes from industrial
wastes
• – Bioleaching does not produce sulphur
dioxide
• – Can often be used in situ
• Potential problem:–Production of sulphuric
acid
Large-scale Production Techniques
• Penicillin
• Made by Batch Culture.
• Penicilium only produces penicillin after it has been
growing for a while.
• It is called a secondary metabolite.
• Fermentation is carried out in a closed or batch
fermenter.
• Microorganisms and a nutrient medium are added to the
fermenter and left for a period of time.
• During the process, nothing is added to or removed
from the fermenter.
• The penicillin is separated from the mixture at the end.
• Temperature is controlled and nutrients are usually
depleted at the end.
• Enzymes can also be manufactured using
a batch culture.
• Eg. Protease for washing powders, which
“react” in the wash with organic stains
• Bacteria provided with a carbon-source
(usually CO2) and a nitrogen-source
(usually ammonia).
• After fermentation the culture is heated to
kill the cells.(The enzyme can withstand
high temperatures)
• The mixture is then filtered and the
enzyme comes out in solution, leaving the
dead cells behind.
Mycoprotein
Continuous Culture
• Fermentation of a fungus, Fusarium, is carried
out in an open fermenter.
• Nutrients are added and product removed at a
steady rate throughout the process.
• Maintains the microorganisms at the exponential
phase of growth.
• Important to monitor pH, temperature and
oxygen concentration as well as levels of
nutrients and product.
• All of these should be kept constant.
Comparing Batch and Continuous
culture
•
•
1.
2.
3.
4.
5.
In general, batch culture methods have certain advantages
over continuous culture.
These are:
easy to set up
the environmental conditions are relatively easy to control
the types of vessels used can be used for different
processes at different times
if the culture becomes contaminated, it is only one batch
that is lost
the level of nutrients drops, which can create the conditions
necessary for the microorganism to manufacture secondary
metabolite such as penicillin
Some advantages of continuous culture are:
1. carried out in smaller vessels, given that
the microorganisms are maintained in
the exponential phase and productivity is
therefore high
2. the high productivity for biomass and
intra- and extra-cellular enzymes is more
cost effective
Disadvantages of continuous culture are:
• microbial growth, clumping of cells and
foaming can tend to block up inlet pipes
• it can be difficult to control all the
environmental factors – if they are not
controlled adequately, there can be a
considerable amount of waste
• it is not possible to create the low-nutrient,
high-stress conditions under which
secondary metabolites such as penicillin
are produced
• Antibiotic – a chemical substance,
produced by a microorganism (bacterium
or fungus) which will inhibit the growth or
replication of other microorganisms.
• Antibiotics bring about their effects in a number
of ways :
• inhibiting protein synthesis – interfering with
transcription or translation.
• interfering with the synthesis of bacterial
cell walls – only effective when the bacteria are
growing.
• interfering with the functioning of the cell
membrane – the bacteria will lose it’s ability to
control the uptake or removal of water and other
molecules.
• inhibiting enzyme activity – this will disrupt
metabolism.
The mode of action of penicillin
on bacteria
• Penicillin is described as a broad spectrum
antibiotic
• It works by interfering with the synthesis of new
cell walls.
• It inhibits the enzymes involved in the synthesis
of cross-links between the peptidoglycan
polymers in bacterial cell walls.
Porins are large
protein channels,
which allow passive
diffusion
Gram positive bacteria stain purple with Crystal Violet dye, and gram
negative
ones stain pink with safranin stain. Named after a Danish bacteriologist,
Hans Christian Gram, and divides bacteria into two broad groupings
• Viruses do not have any form of cell
structure or metabolism – hence,
antibiotics are ineffective against viruses.
• They replicate only within the living host
cells, and make use of the living host cell’s
transcription and translation mechanisms.
The absence of any sort of cell wall
means that penicillin has no effect on
viruses.
The causes and effects of antibiotic resistance
• Bacteria are genetically variable. These
variations occur by natural mutation giving
rise to new alleles of genes.
• Natural selection can change the
frequency of these alleles in the
population so that most of the bacteria in
the population are resistant, by the
following steps:
• some bacteria have alleles of genes which give
resistance to a particular antibiotic
• an infection leads to treatment of the infected
person with an antibiotic
• the antibiotic will kill susceptible bacteria, but
resistant bacteria will survive
• only the resistant bacteria will reproduce,
resulting in an increase in the frequency of the
bacteria that are resistant to that particular
antibiotic. An example of directional selection.
• people infected in the future are infected by
bacteria more likely to carry the alleles for
resistance
• The alleles of genes that cause resistance
arise for the first time by mutation and are
often located on plasmids, which means
that they can rapidly spread from one
bacterial species to another since
plasmids are naturally exchanged between
species. Bacteria of different species may
come together, and the plasmids will
transfer either by direct contact or through
a special channel.
• The plasmids may also contain a number
of different antibiotic resistance genes so
that species can suddenly acquire
resistance to a number of antibiotics when
before they had none.
Enzyme immobilisation
• the attachment of enzymes to insoluble
materials, which then provide support for
the enzymes.
• This allows enzymes to be held in place
throughout the reaction, following which
they are easily separated from the
products and may be used again.
Different ways to
immobilize
enzymes
The process of immobilization in alginate
beads involves the following stages:
– An enzyme is mixed with a solution of sodium
alginate
– this mixture is dripped (usually through a
syringe) into a solution of calcium chloride
– the sodium ions are displaced by the calcium
ions, resulting in the formation of hard,
insoluble beads of calcium alginate, in which
are trapped the molecules of amylase
– the alginate beads are left to harden further,
and then rinsed, and stored for future use.
Example 1: Using
immobilized
lactase to prepare
lactose free milk
for people with
lactose intolerance
• Example 2. Producing Maltose from starch
• The beads (having been made to include amylase
enzyme) are placed in a suitable container to create a
column of beads.
• A suspension of starch can then be trickled down the
column and the product is collected in a beaker.
• a solution of maltose, without any starch will be present
in the beaker
• By hydrolysing the starch this way, there is no
contamination of the product with enzyme – and the
amylase remains in the beads, which can then be used
again.
advantages of immobilisation:
• enzyme can be recovered after use using
a very coarse filter rather than a molecular
filter
• enzyme does not contaminate product
• immobilisation may enhance stability
(thermostability or pH-stability) of the
enzyme molecule as it is supported
• substrate can be easily passed through
the enzyme several times
Biosensors and dipsticks
• Industrially produced enzymes are used to test
for the different levels of substances in the body.
• Glucose oxidase is used by diabetics to test their
blood glucose concentration.
• It is immobilised and stuck on the end of a
dipstick.
Dipsticks.
These ones
test urine for
Glucose
(diabetes),
Blood (kidney
problems),
Protein (liver
problems),
PH (metabolic
imbalance).
• When it comes into contact with glucose it
oxides it to gluconolactone and causes a
colour change.
• The more glucose the darker the colour.
• Biosensor - a device which makes use of
a biological molecule to detect and
measure a chemical compound.
• Most people now use a biosensor which
detects an electric current genetrated
during this oxidation reaction, which is
read by a meter, and displays blood sugar
levels.
Biosensor and
test-strips
Revision: Antibodies
In our immune system one type of lymphocyte is a B
Lymphocyte.
This divides by mitosis to create copies of itself.
Sometimes it divides to become a Plasma Cell,
which will produce antibodies in response to a
foreign chemical (antigen).
When a foreign cell invades an organism, there are a
lot of different types of antigen on its surface
membrane, so the B lymphocytes divide to make lots
of different plasma cells to counteract each different
antigen.
Monoclonal antibodies
Identical antibodies produced to be effective
against a single, specific antigen.
• Problem with producing them – B
lymphocytes that produce antibodies don’t
divide, and B lymphocytes that divide
(making plasma cells) don’t produce
antibodies!
HAT medium is
hypoxanthine
aminopterin thymidine.
Only hybridomas
survive. Myeloma dn
normal cells die.
• Plasma cells are fused with cancerous
cells which go on dividing indefinitely.
• This formed a hybridoma which divides
and secretes antibodies.
Using monoclonal Antibodies
• 1. Pregnancy Tests
• The monoclonal antibodies are made that
bind with the human hormone human
chorionic gonadotrophin. (hCG)
• The antibody is attached to a dipstick.
• The dipstick is dipped into a urine sample.
• Any hCG in the urine will bind to the
antibody and will be carried up the stick
with the urine.
• Another antibody is made which will bind
with the hCG-antibody complex.
• This is placed further up the stick and
immobilised.
• As the hCG-antibody reaches the
immobilised antibody it binds and a pink
colour forms. (or blue, depending on the
brand)
As a test, at the top of the stick a different
antibody has been imobilized. This comes
from cells of a different animal (often a
goat), which will naturally react (by binding)
to the antibody at the bottom of the stick.
It will only bind with mouse antibodies that
have NOT got the hCG hormone attached
to it, and again produces a coloured band.
One band: Negative, as there was no hCG.
Two bands: Positive, as some of the mouse
antibody has hCG bount to it.
Monoclonal antibodies in diagnosis
and treatment