Week 2 Flashcards
(147 cards)
1
Q
What are the key features of prokaryote chromosomes?
A
Usually just 1, circular, from 0.5 to 14 Mbps in size –> prokaryotes are haploid
2
Q
What are the key features of extrachromsomal DNA?
A
Generally non essential, small self-replicating molecules that confer adaptive advantages –> plasmids
3
Q
Where is prokaryote chromosome?
A
Their chromosome is free in the cytoplasm but very densely packed. The region of the cytoplasm that contains this genetic material is called NUCLEOID.
4
Q
How can the size of prokaryote chromsomes differ?
A
A classic example of small genome is Mycoplasma genitalium (human pathogen), which has a 580 kb genome
Symbiotic bacteria - Carsonella ruddii, which lives off sap-feeding insects, has taken the record forsmallest genomewith just 159,662 bp
Among the biggest genomes Sorangium cellulosum, with 13 Mb
5
Q
What are operons?
A
Genes are usually close to each other which are under under the control of a single regulatory sequence.
6
Q
What does an operon do?
A
One operon is transcribed into a single mRNA molecule that encodes several proteins (polycistronic)
7
Q
Do porkaryotic mRNAs undergo posttranscritional modifications?
A
Prokaryotic mRNAs do not undergo posttranscriptional modifications (they don’t have introns either)
8
Q
What does the abscence of posttranscriptional modifcations mean for translation?
A
The absence of posttranscriptional modifications, plus the lack of a nuclear membrane barrier allow for transcription and translation to occur almost simultaneously –> Both process are coupled
9
Q
What is the overall coding to noncoding relationship in prokaryotes?
A
Most of the prokaryotic genetic material is coding DNA, unlike in eukaryotes
10
Q
What is the overview of prokaryote operons and transcription?
A
Polycistronic operons (several genes per mRNA)
Genes with no introns
No posttranscriptional modification of mRNA
Transcription and translation coupled in the cell
11
Q
What is the overview of eukaryote operons and transcription?
A
Monocistronic operons (one gene per mRNA)
Genes have introns (non-coding sequences within the gene)
Posttranscriptional modification of mRNA is required for translation
Transcription and translation are uncoupled (mRNAs need to leave nucleus to be translated)
12
Q
What is horizontal gene transfer?
A
Movement of genetic material between two cells that are not necessarily related, as opposed to the transfer from mother to daughter cells, known as vertical gene transfer
13
Q
What is the advanatage of horizontal gene transfer?
A
The prokaryotic genome is extremely adaptable and quickly evolving, thanks mostly to horizonal gene transfer
14
Q
What is the key feature of horizontal gene transfer?
A
HGT is, alongside the occurrence of spontaneous mutations, the main generator of genetic diversity in bacteria that allows them to adapt to new, often hostile environments.
15
Q
What are the 3 types of horizontal gene transfer?
A
Transformation
Conjugation
Transduction
16
Q
How has horizontal gene transfer shown to be a quick driving force in prokaryote evolution?
A
Ability to use new substrates as food sources
Antibiotic resistance
Ability to detoxify harmful chemicals (heavy metals, salts)
Virulence
17
Q
What are landmark evolution stages involving acquistition form distant organisms?
A
Archaea establishing symbiosis and internalising bacteria as the source of eukaryotic organisms)
18
Q
What is transformation?
A
Direct uptake of extracellular “naked” DNA by the bacterial cells
19
Q
Where does the DNA uptaken by transformation come from?
A
Acquired DNA usually comes from nearby degraded cells.
This DNA can correspond to fragments of chromosome or to extrachromosomal DNA such as plasmids.
20
Q
What can happen to the uptaken DNA when in the bacteria?
A
The acquired material can be degraded by the cell and used as building blocks for its own metabolism, or maintained stably, either by integration in the chromosome. Replicating independently from chromosome (only plasmids)
21
Q
What is the name of the physiological state that allows for the acquisition of exogenous DNA?
A
Competence
22
Q
What are the two types of competence?
A
Natural and Induced
23
Q
What is natural competence?
A
Genetically encoded capability to uptake and incorporate exogenous DNA, some prokaryotes have genes encoding the machinery necessary to capture, transport and incorporate into their chromosome.
24
Q
How frequent is natural competance?
A
More than 80 species including both Gram-positive and Gram-negative bacteria are naturally competent
25
When was natural competance first described?
This phenomenon was firstly described by Frederick Griffith in 1928, after observing the transference of virulence between Streptococcus pneumoniae strains.
26
What was Griffith's experimental setup which natural competance was first described?
Griffith’s experimental setup, led him to the existence of the transformation principle: non-virulent Streptococcus pneumoniae (a rough strain) could convert to a virulent pathogen when co-inoculated with a heat-killed virulent S. pneumoniae (a smooth strain) and injected into mice.
27
What is the reasoning behind Griffiths experiments?
The absence of disease symptoms after monoculture inoculation due to the lack of a specific virulence factor in the rough strain and the heat inactivation in the smooth strain. However, in addition to the ability of the mixed culture to kill the mice, Griffith was also able to re-isolate live virulent (smooth) bacteria from the infected animals
28
What is induced competence?
Technique carried out in laboratory settings to introduce DNA in non naturally competent cells. This is one of the essential techniques in molecular biology
29
What is required for induced competence?
Requires the destabilization of the bacterial membranes
30
What are 2 techniques for induced competence?
By treating with chemicals (such as CaCl2): chemically competent cells --> transformation by heat shock
By applying electric shock: electrocompentent cells --> transformation by electroporation
31
What is conjugation?
Mechanism of DNA transfer involving direct cell to cell contact via the formation of a structures called conjugal pili
32
What is the overview of conjugation?
Conjugation is the main transfer mechanism for plasmids.
DNA is transferred unidirectionally from one donor cell (F+) to a recipient cell (F-)
33
When was conjugation first described?
This phenomenon was first described by Joshua Lederberg and Edward Tatum in 1946
34
How was conjugation discovered?
They discovered that E. coli mutant cells impaired in the production of some essential nutrients (auxotrophs) recovered the ability to produce them (prototrophs) after being in direct contact with other E. coli cells possessing the biosynthetic capabilities they were missing
35
How did they prove it wasnt through transformation?
Naked DNA didn’t have the same effect, and it was required that the cells were in direct contact.
36
What is the mechanism for conjugation?
The donor cell produces a pilus that attaches to receptors in the recipient cell.
After contact, the pilus retracts, bringing the cells closer and establishing a relaxosome bridge.
The plasmid is nicked at specific location called origin or transference (oriT) and one of its strands transfers through the relaxosome. It starts replicating in the donor cell. Once in the recipient cell, the other strand replicates too.
After reconstitution of the plasmid, the recipient cell becomes a potential donor (F+) .
37
What are plasmids?
Plasmids are extra chromosomal DNA molecules that are physically separated from chromosomal DNA and can replicate independently. They are mostly circular, double stranded DNA molecules. Very diverse in size.
38
What are episomes?
When a DNA plasmid is integrated into the chromosome
39
What are the key function of plasmids?
Plasmids are not essential, but often carry genes that confer multiple advantages to their hosts
40
What are examples of key function plasmids bring to their hosts?
Ability to conjugate (F factor in E. coli).
Antibiotic resistance and/or biosynthesis.
Heavy metal resistance.
Nitrogen fixation and nodulation (symbiosis with legumes).
Utilization of uncommon substrates as nutrients.
Degradation of toxic molecules.
Virulence.
41
What happens if a bacteria has several types of different plasmids?
Several different plasmids can coexist in the same cell, unless they have the same replication machinery, in which case they are mutually exclusive and they are classified as part of the same incompatibility group
42
What are the key section of a DNA plasmid?
origin of transference (oriT)
origin of replication
Cargo (antibiotic resistance gene / virulence/ degradation / biosynthesis genes)
Mobilisation genes
43
How diverse is conjugation?
Conjugation can happen between cells of different species, or even different kingdoms of life.
44
What is an example of cross kingdom conjugation?
Agrobacterium tumefaciens, a plant pathogen responsible for crown gall in over 140 species of plants.
45
How does Agrobacterium tumefacians cause crown gall?
A. Tumefaciens contains a virulence plasmid (Tumor inducing or pTi plasmid). This plasmid contains all the genes necessary for conjugation and a region called T-DNA, that contains genes for the synthesis of plant hormones and nutrients for the bacterium.
46
What happens when the Agrobacterium tumefacians plasmid is introduced to the plant?
After penetrating the plant through a wound and establishing a conjugation pilus, the plasmid excises the T-DNA and it is transferred to the plant cell nucleus, where it integrates in the plant genome.
The T-DNA is then expressed and the resulting hormones stimulate cell growth and tumour formation.
47
What is the use of Agrobacterium conjugation into plants?
The ability of Agrobacterium to introduce DNA in plants has been harnessed for biotechnology applications, and is a commonly used tool in labs to genetically manipulate plants
48
How can conjugation be used on bacteria?
Interspecies conjugation is a technique routinely used in the lab to transfer plasmids between different bacteria, especially to those that are difficult to transform.
49
How are plasmids and conjugation useful for experiments?
Thousands of artificial plasmids have been generated for multiple molecular biology and biotechnology purposes (gene and protein expression, mutant generation). Carrying human-designed cargo and selectable markers (e.g antibiotic resistance genes) to select for cells containing the plasmid
The manipulation and transfer of plasmids are also basic molecular biology and biotechnology techniques
50
Whats transduction?
Transduction can happen when a phage or prophage initiate a lytic cycle and bacterial DNA is packed in the newly formed viral particles. This new particles can then infect other bacteria and transfer them this DNA.
51
What is the advantage of transduction?
This mechanism doesn’t require direct contact between the cells, as it is the phage the one that carries the DNA
52
What happens to the bactrial DNA?
Phages infect bacteria and inject their own genetic material into the bacterial cytoplasm
53
What are the different outcomes of the phages and bacteria DNA which has been inserted?
The viral genetic material is recognized and destroyed by bacterial immunity systems.
The phage genome hijacks the cell machinery and starts multiplying, eventually killing the cell and releasing all the new phage particles --> lytic cycle.
The phage genome integrates in the bacterial chromosome becoming a prophage and stays dormant or expressing at low level --> lysogenic cycle.
54
What happens to the Lytic cycle?
Phage attaches to a host cell and injects its DNA
Phage DNA circularises
Certain factors induces lytic cycle
New phage DNA and proteins are synthesised and assembled into phages
The cell lyses, releasing phages
55
What happens in the Lysogenic cycle?
Phage DNA integrates into the bacterial chromosomes, becoming a prophage
The bacterium reproduces normally, copying the prophage and transmitting it to daughter cell
Many cell divisions produce a large population of bacteria infected
Occasionally, a prophage exits the bacterial chromosomes initiating a lytic cycle
56
What are the different transduction types?
Generalised transduction
Specialised transduction
Lateral transduction
57
What is generalised transduction?
Transfer of DNA from any part of the host genome to the recipient cell
58
What is specialised transduction?
Transfer of a few specific sets of genes between cells
59
What is lateral transduction?
Seen in S. aureus, similar to specialised transduction but capable of transferring more host DNA at much higher frequency rates
60
When does generalised transduction occur?
Phages package any bacterial DNA (chromosomal or plasmid) and transfer it to another bacterium
61
What phages does generalised transduction occur in?
This sort of transduction is typical of pac-type phages (e.g. it is carried out by Salmonella phage P22 – a pac phage – this was the first mechanism of phage-mediated gene transfer identified)
62
How can generalised transduction occur?
These phages recognise pac sequences within their DNA that indicate the end of their genome and aids them during its packaging in the viral capsid. However, they sometimes recognise similar sequences in the host genome (pseudo pac sites) and pack that instead of their own genome.
63
What is the frequency of generalised transduction?
It is estimated that 1/100-1/1000 of viral particles contain host DNA instead of phage DNA --> transducing particles
64
When does specialised transduction occur?
Occurs when prophages exit the lysogenic cycle and enter lytic cycle, and only selected sets of genes are transferred.
65
Where was specialised transduction discovered?
This was the second system of bacterial transduction discovered, and it was reported in the λ phage from E. coli.
66
What causes specialised transduction to occur?
When the prophage resumes the lytic cycle it excises its DNA from the host genome to start replicating. This excision is sometimes aberrant, generating hybrid molecules that have part of the host DNA.
67
Why is specialised transduction called specialised?
This mechanism is called “specialised” because phages integrate at specific locations of the host genome (attb sites in the case of λ ) and only the contiguous genes are carried
68
What is a difference between generalsied and specialised transduction?
This mechanism of transduction is less frequent and powerful than the generalised one.
Transducing particles are hybrid
69
When does lateral transduction occur?
A variation of the specialised mechanism, allows transfer of larger bits of host DNA.
Described for the first time in 2018 in Staphylococcus aureus
70
What happens in lateral transduction?
Prophage excision occurs late in the lytic cycle, well after DNA replication and packing have started.
Viral DNA starts being packed while still attached to the chromosome and large stretches of the host DNA contiguous to the prophage location are packed inside successive viral capsids.
In parallel, several rounds of replication are taking place at the prophage location, magnifying the effect
71
What are the advantages of lateral transfer?
This leads to the production of high titres of transducing particles, and this mechanism can transfer more genes and at higher frequencies than generalised and specialized transduction.
Transducing particles can be hybrid or carry only host DNA
72
What are the potential applications of transduction?
Use of capsids as delivery vehicles.
Use of the viral packing system to make DNA libraries.
Use of transduction to transfer DNA between cells.
Engineering of the integration machinery of phages into plasmids, to make them able to stably integrate in the recipient cell
73
What is a genome?
The total complement of genetic information of a cell
74
What is genomics?
The discipline of mapping, sequencing, analysing and comparing genomes
75
How big is the bacterial genome?
Typically, a bacterial chromosome ranges from 0.5 to 14 Mbp size
Direct proportion - genome size increase also increase in gene number
Bacterial genomes encode approx. 100 ORFs (Open Reading Frames) per 1 Mbp → each ORF is ≅ 1000 bp.
76
What is core genome?
All genes that are shared by all strains of a given species
77
What is pan genome?
Includes the core genomes + genes not shared by all strains of a given species
78
How large is the pan genome?
Its size is difficult to define because it increases as the genomes of more strains of a species are sequenced
79
What genes made up the pan genome but not the core genome?
Encode genes that give the strain accessory capability: virulence, biodegradation, antimicrobial resistance
80
How can horizontal gene transfer impact bacterial genomes?
Horizontal gene transfer contributes to the evolution and diversity of the genomes
HGT confers to the cell different traits that can be key for survival in different environments (antimicrobial resistance, metabolic capacities)
Different genes/genetic elements can be initially acquired by HGT and then be vertically transmitted
81
What is mobilome?
The sum total of all mobile genetic elements in a genome,
Including:
Plasmids
Prophages (integrated virus genomes)
Other mobile elements: integrons, insertion sequences and transposons
82
What is the function of mobile genetic elements?
Mobile genetic elements promote genome evolution
83
What is the origin of CRISPR and restriction modification systems?
Bacteria posses several “weapons” to defend from from exogenous DNA attack (typically phage DNA)
84
What is the restriction process?
Bacteria can destroy double-stranded exogenous DNA at specific sites by the activity of Restriction Endonucleases (Restriction Enzymes: RE)
85
What is the modification process?
Host has to protect its own DNA from restriction enzymes attack. Host modifies its own DNA, typically by methylation of nucleotides where RE cut
86
What is the overview of restriction enzymes in bacteria?
Part of the Restriction Modification System
Are specific for double-stranded DNA. Single-stranded viruses or RNA viruses CANNOT be cut by RE.
Recognise a specific sequence of nucleotides (Recognition Sequence)
Cleaves ds DNA at or near the recognition sequence
Recognition Sequences are usually palindromes of 4–10 bp long
87
What is a palindromic sequence?
A palindromic DNA sequence in double stranded DNA: when a sequence read in a direction matches the sequence on the complementary strand read in the same direction
88
What is the sequence and cut of EcoR1?
GAATTC ---> GA ATTC --> Sticky end
CTTAAG CTTA AG
89
What is the sequence and cut of EcoRV?
GATATC ---> GAT ATC --> Blunt end
CTATAG CTA TAG
90
What is the overview of restriction enzymes in gene editing?
A very important tool in molecular biology, biotechnology
They recognise a specific sequence of nucleotides and precisely cut double stranded DNA at or near the recognition sequence. They are called “molecular scissors”.
Commercially available.
Are widespread used in molecular cloning
91
What is molecular cloning?
Is the movement of a desired gene or DNA fragment (the insert) from their original source to a small genetic element (the vector). This results in recombinant DNA.
92
What is the use of molecular cloning?
When the recombinant vector is transfer into an appropriate host, the cloned DNA is replicated and ‘amplified’.
This procedure is widely used in molecular biology and biotechnology with many different purposes
93
What is the process of molecular cloning?
1 - Cut With restriction Enzymes:
- DNA Source
- Vector
2 - Put together, Insert, cut vector and add ligase
3 - Introduce recombinant vector in a host
94
Where do most elements in molcular cloning come from?
Most of the elements used in this process have been taken and adapted from nature:
Vectors, restriction enzymes, ligases (come from bacteria and phages), transformation
95
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
96
What is CRISPR and where is it present?
Prokaryote defence mechanism. Present in 50% of Bacteria and 80% of Archaea.
‘immune system’ used by microorganisms to defend themselves against invading viruses and other mobile genetic elements.
It also helps the cell to maintain stability and integrity of its genome.
97
How does CRISPR work in the genome?
Contains many different segments of foreign DNA called Spacers
Spacers are alternated with identical repetitive sequences
Spacers correspond to pieces of foreign DNA that have previously invaded the cell
98
What happens to the CRISPR region when transcribed?
The CRISPR region is transcribed into a long RNA molecule
This long transcript is then processed into segments by CRISPR-associated (Cas) proteins, converting the long RNA molecule into segments of small RNASs called CRISPR RNAs (crRNAs)
99
What does the CRISPR RNA do when produced?
If one crRNA base-pairs with an invading nucleic acid, the invading nucleic acid is destroyed by the endonuclease activity of a Cas protein.
100
What are the applications of CRISPR?
CRISPR/Cas9 system has being used for Genome Editing of diverse organisms
Used to cut specific DNA sequence of the genome of virtually any cell
The native system has been simplified and a synthetic guide RNA (sgRNA) is design to target the sequence to be edited
Genes encoding for the sgRNA and Cas9 are often cloned into a plasmid and delivered into the target cell
101
What are examples of CRISPR being used?
Genome-wide CRISPR screen identifies host dependancy facotrs for Influenza A
Modify Saccharomyces cerevisiae for bioproduction of useful chemicals
Produce parthenocarpic tomato plants
102
Who and when was the nobel prize won in chemistry for the application of CRISPR?
Emmanuelle Charpentier and Jennifer A Doudna in 2020
103
What is the primary form of reproduction in prokaryotes?
Binary fission is a common way for bacteria and archaea (and also some eukaryotes e.g. Euglena) to reproduce
104
What is binary fission?
Asexual reproduction, where a parent cell divides to produce two identical daughter cells
105
What are the basic steps of binary fission?
In preparation for binary fission, DNA replicates and the cell grows.
DNA migrates to the opposite poles the cell, and a septum forms in the middle
A new cell wall forms at the septum, and the cell separates into two daughter cells
106
What major structures of involved in rod shaped cell division?
Nucleoid occlusion
Nucleoid
MinC gradient
Z ring
107
What is the final step in coccoid shaped cell division?
The final stage of cell division may involve cells ‘snapping off’ each other
108
How do eukaryotic microbes divide?
Eukaryotic cell division involves mitosis, the segregation of pairs of chromosomes in the nucleus.
109
What is another form of eukaryote microbe division?
Some eukaryotic microbes have very complex life cycles involving different forms, as well as budding.
110
What is required in budding?
A septin ring in which a growing bud (daughter cell) forms, this then develops into a myosin ring during metaphase. During anaphase chromsomes are pulled apart. Mitioic exit is when the septin splits
111
What is unlimited population growth of microbes like?
Unlimited population growth is exponential. If it is assumed that growth occurs without limitations
112
What is growth rate directly proportional to?
The growth rate, or rate of increase in populations numbers/biomass is proportional to the population size at a given time.
113
What are examples of linear growth?
Plant growth
114
What are exmaples of expotential growth?
epidemics eg covid and (wild)fire
115
How quickily can a single E.coli divide?
Under ideal conditions around every 20 minutes
116
What does a single E.coli dividing every 20 minutes quickily become?
it would take a day and a half for a super-colony to weigh the same as the entire planet
In several days the biomass would weigh more than the entire solar system
117
What can limit prokaryote cell division?
Limited nutrients
Predation (viruses or organisms higher on the food chain)
Space
118
What is the name of bacteria are in an unlimited environment, they will divide at a constant interval?
The generation or doubling time (length of time it takes for the cell population or biomass to double)
119
What factors can impact the generation or doubling time?
The species
The growth medium
The temperature
The pH
120
How can you calculate the number of bacteria after a certain number of generations dividing?
Starting with any number of organisms (N0), the number of organisms after n generations will be N0 x 2n.
121
What are the key features of batch culture?
Batch culture is a closed culture system which contains a limited amount of nutrients.
122
What are the names of the growth phases?
Lag phase
Exponential (log) phase
Stationary phase
Death phase
123
What happens during the lag phase?
When cells are transferred from an old to a new environment, time is needed to respond to their environment and to express specific genes and synthesise components needed for rapid growth.
124
Why is there a lag phase?
Cells may be damaged
C, N or energy sources may be different in the new environment.
Cellular machinery needs to be turned on (RNA transcribed, enzymes must be synthesised, etc
125
What can impact the length of the lag phase?
The length of lag phase varies e.g. due to media changes, temperature changes etc.
Cells grown in complex medium which are transferred to minimal medium will have a longer lag phase.
126
What are the key features of early exponetial phase?
Exponential growth is balanced growth where the cell components are synthesised at a constant rate relative to each other.
Cells are growing and dividing at the maximum rate possible based on the media, and the growth conditions provided.
Cells are often at their largest at this stage.
127
What is key for bacterial metabolism in early exponential growth?
Primary Metabolites (Directly involved in growth and reproduction):
E.g. enzymes such as proteases, amino acids, vitamins, nucleosides
128
What is downshift and upshift?
Downshift-moving cells from a good carbon source to a poor carbon source
Upshift-moving cells from a poor carbon source to a good carbon source
129
What are the key features of late exponential phase?
At this point the rate of doubling slows.
New growth phase dependent genes are expressed
Some species can also detect the presence of other cells, by sending and receiving signals (quorum sensing).
130
What are the key features of the stationary phase?
Cells numbers have stopped increasing (no new cell growth) due to the lack of a key nutrient, or the build up of waste and toxic products eg o2 radicals by-product metabolism
For bacteria in complex media this is generally when the cell density rises above 109 cells per ml.
Eukaryotic microbes, eg protozoa enter stationary phase at lower population densities, at around 106 cells per ml.
Microbes change their physiology at this stage, if they did not they would be vulnerable
131
What are physiological changes seen in the stationary phase?
Some can differentiate into resistant spores.
Some bacteria, e.g. E.coli reduce their cell size, minimizing the volume of the cytoplasm in relation to the volume of the nucleoid.
Stress resistant enzymes are produced to handle oxygen radicals, to protect DNA and proteins.
132
What impacts do stationary phase have with its envrionment?
Cells in stationary phase can become more resistant to heat, osmotic pressure, pH changes and other stresses.
Stationary (secondary) phase metabolites (generally have an ecological function):
133
What are the key features of the death phase?
Without new nutrients cells will eventually die due to the build up of toxic by-products.
The death rate is the rate at which the cells die
Knowing death rates is important in food preservation and antibiotic effectiveness.
134
How would you plot cellular division?
If cell number in the population is plotted onto a graph, you would get an exponential curve.
But it is often easier to plot the data on a logarithmic scale on the y-axis which then gives a straight line
135
What is the formula for the relationship between specific growth rate and doubling time?
mew (u) (specific growth rate) = natural log of 2/ doubling time (td)
136
What is a second forumla for working out specific growth rate and doubling time relationship?
mew (u) (specific growth rate) = (natural log of N1 - natural log of N0) / (t1 - t0)
137
What are the three main ways microbial cells can be grown in bith the loboratory and on an industrial scale?
Batch culture
Fed-batch culture
Continuous culture
138
How can microbial growth be measured?
Growth can be measured by OD (optical density), cell number or dry cell weight
139
What are the key features of batch culture?
Transfer of a small portion of a culture into new culture medium, resulting in growth and an increase in biomass
Closed system of cultivation (except possibly for aeration)
Minimal chance of contamination
LOW PRODUCT YIELD
Used for making cheese/yoghurt
Common in lab experiments
140
What is a case study for the use of batch cultures?
Penicillin production
Use large industrial fermenters
Add Penicillium mold and nutrients
Grow for 6-8 days
Separate the penicillin from the mold and purify
141
How can you optimise growth of microbes in batch cultures?
Can alter different parameters to improve growth
Oxygen availability (anaerobic or aerobic growth)
Growth media
Growth media can often consume much as 50% of all costs
pH
Temperature
142
What are the key features of fed-batch cultures?
Same as batch culture except at certain intervals particular nutrients are added
Semi-closed system (greater chance of contamination)
Prolonged exponential phase
Used more in biotechnology
Higher yields of active product
143
What is the consequence of adding more nutrients in fed-batch cultures and continuous cultures?
If more nutrients are being added then this increases the cell mass, this is linked to cell division, thus the dilution rate is directly related to growth rate
However there comes a point where the dilution rate is so fast, that cells are washed out faster than they can be replaced by division, thus biomass reduces
144
What can limit the amount a culture can be batch fed?
Build up of toxins can limit how many times a culture can be batch-fed
145
What are the overview of continuous cultures?
These are open culture systems where new fresh medium is constantly added to a culture, and a equal volume of culture is constantly removed.
Microbial cultures can be kept in exponential phase at a constant cell mass for extended periods of time (called steady state growth).
146
What is the dilution rate of continuous cultures?
Dilution rate is related to growth rate, more nutrients which are received as a result of increasing flow rate, the faster the cells can replicate, which decreases generation time
147
What is the process of continuous culture?
Addition of media, base and large volume culture
Stirrer in the large volume culture
Then removal of media and cells which can then remove produce to be purified
