How Cellular Information is Altered Flashcards
HOW CELLULAR
INFORMATION IS ALTERED
Mutation and Selection
Natural Mechanisms for Gene Transfer and Rearrangement
Genetically Engineering Cells
Genomics
We can alter cells by using mutation or genetic engineering.
HOW CELLULAR
INFORMATION IS ALTERED
____________ is the purposeful transfer of DNA from one type of organism to another.
Genetic Engineering
_________ is subjecting the cells to stress, causing changes in the genetic make-up.
Mutation
___________ = mistakes in the genetic code (can arise from replication and/or damage)
Mutations
____________ = organism with a genetic mutation
Mutant
_________ = the organism without the genetic organism
Wild type
______________ = genetic construction of an organism
Genotype
_________ = characteristics expressed by an organism.
Phenotype
__________ = usually refers to transcription + translation + post-translation processing.
Expression
If the mutation is in the active site, there may be some _________ consequences.
enzyme activity
If the mutation changes the amino acid to a ________, the resulting _______ will be ________ and probably ___________.
stop codon, protein, truncated, not active
If the amino acid is the same as before the mutation there is _____________
no consequence
If the amino acid is different, but not in the ________ of the active site, there _________
region, may be no consequences.
confers upon the mutant an advantage for growth, survival or detection under a set of environmental conditions that the wild type does not have
Selectable mutation:
Antibiotic resistance
Ability to grow on toluene
Inability to produce lysine
Ability to produce bioluminescence
Ability to produce more of an enzyme
Inability to grow at higher temperatures
SELECTION
____________ mutations per cell conversion
10^-3 - 10^-9
10^-6 = ________________
1 mutation/1,000,000 divisions
_________: chemicals, radiation
Mutagens
Lots of growth (i.e. lots of divisions)
INCREASE MUTATION RATES
WHY DO WE WANT TO
INCREASE MUTATIONS?
We want a cell to develop specific characteristics that are advantageous for us.
_____________: uptake of free DNA by a cell. The cell membrane has to be permeable to DNA.
Transformation
_____________: DNA is carried into the call in a phage.
Transduction
_________: Cell-to-cell transfer of DNA.
Conjugation
Also called mating.
Conjugation
Using ___________, engineers and microbiologists were able to increase penicillin from 0.001 g/L to 50 g/L.
mutation and selection
Using natural mechanisms to purposefully manipulate DNA. The DNA is manipulated outside of the cell, and then sent into the cell.
GENETIC ENGINEERING
__________: enzymes that cut DNA at specific sequences. Different enzymes will cut at different sequences.
Restriction enzymes
______________: A method to detect what sizes of DNA a sample contains.
Gel electrophoresis (Southern Blot)
______________: A process used to make many copies of a piece of DNA.
Polymerase chain reaction (PCR)
_____________: self replicating, circular piece of DNA that can survive in a cell.
Plasmid
________________recognition sequence cuts long DNA more frequently and produces smaller DNA fragments than a restriction enzyme with a six-nucleotide recognition sequence.
A restriction enzyme with a four-nucleotide
Any given __________ occurs in DNA, on average, at a distance of 256 (4^4)nucleotides.
four nucleotide long recognition site
Any given _________________ occurs, on average, at a distance of 4096 (4^6) nucleotides.
six nucleotide long sequence
Any given __________________ occurs, on average, at a distance of 65536
(48) nucleotides.
eight nucleotide long sequence
allows scientists to extract and analyze bits of microbial DNA from samples, meaning they don’t need to find and grow whole cells.
PCR
is an essential element in DNA fingerprinting and in the sequencing of genes and entire genomes.
PCR
Basically, it’s like a technique to photocopy pieces of DNA. In a matter of a few hours, a single DNA sequence can be amplified to millions of copies
PCR
lets scientists work with samples containing even very small starting amounts of DNA.
PCR
The technique makes use of the DNA repair enzyme polymerase. This enzyme, present in all living things, fixes breaks or mismatched nucleotides in the double- stranded DNA helix. These breaks or mismatches could cause genes to malfunction if left unfixed.
PCR
Polymerase uses the intact half of the DNA molecule as a template and attaches the right nucleotides, which circulate constantly in the cell, to the complementary nucleotide at the site of the break. (DNA consists of two strands of nucleotide bases, which are represented as A, G, C, and T. In the laws of DNA base-pairing, A joins with T and G with C.)
PCR
Not all polymerases are created equal, however. Many fall apart in high heat.
PCR
PCR was developed in ______ following the discovery of an unusual heat-loving bacterium called ________ in a hot spring in __________
1985, Thermus aquaticus, Yellowstone National Park.
This bacterium’s polymerase, dubbed Taq, does its job of matching and attaching nucleotides even in the high heat generated by the successive_________ cycles required during PCR.
“photocopying”
______ made PCR possible.
Taq
_________: changing conditions - transient (S, X, growth rate), high initial substrate, different phases of growth.
Batch
________: steady-state, constant low concentration of substrate, constant growth ratethat can be set by setting the dilution rate (ie. the feed flow rate).
Chemostat
__________ is more efficient.·
Chemostat
____________ is more common.
Batch
CHOICE OF CONTINUOUS VS
BATCH PRODUCTION
Productivity
Flexibility
Control
Genetic stability
Operability
Economics
Regulatory
_________: rate of product per time per volume. Chemostat is better for growth-associated products. Wasted time in batch process.
Productivity
____________: the ability to make more than one product with the same reactor. Batch better.
Flexibility
___________: maintaining the same conditions for all of the products produced. In theory, the chemostat is better, steady state. In reality???
Control
____________: maintaining the organism with the desired characteristics. Chemostat selects for fast-growing mutants that may not have the desired characteristics.
Genetic stability
___________: maintaining a sterile system. Batch better.
Operability
____________: validating the process. Initially, many process batch, too expensive to re-validate after clinical trials.
Regulatory
Consider the production of a growth-associated product (like cell mass) in _________.
suspension culture
__________ of a chemostat is detrimental to engineered organisms.
Selective pressure
______ is more mechanically reliable.
Batch
________ system is more more flexible.
Batch
SPECIALIZED REACTORS
Chemostat with recycle
Multistage chemostat
Fed-batch
Perfusion
_________ under the control of an inducible promoter
Recombinant product
________ at the same rate if the recombinant product is not expressed.
Recombinant strain and wild type grow
If the __________is expressed, the _____________ grows much slower.
recombinant product, recombinant strain
First chemostat is fed with a non-inducing growth substrate, allowing the recombinant strain to be produced.
MULTISTAGE CHEMOSTAT
The effluent from the first chemostat feeds a second chemostat that is fed inducer, and the product is produced.
MULTISTAGE CHEMOSTAT
Note: new recombinant cells are continually added to the second chemostat not allowing take-over by a fast growing mutant.
MULTISTAGE CHEMOSTAT
_________ reactors gain some advantages of a CSTR, retain some disadvantages of batch
Fed-batch
Reduces substrate inhibition or catabolic repression, allows for high conversion, and the extension of stationary phase.
Fed-batch
______ nature usually leads to higher operation cost and batch variability.
Semi-batch
________ cultures are started as batch cultures and grown to an initial cell concentration
X, after which fed-batch operation begins.
Fed-batch
Substrate is consumed at the same rate it is added.
QUASI-STEADY STATE
Usually, fed-batch cultures are taken through many feedings cycles, with each feeding cycle followed by a harvest cycle during which the volume is drawn back down to V0 and the cycle begun again.
REPEATED FED-BATCH
PERFUSION CULTURE
Animal Cell culture
Constat medium flow
Cell retention
Selective removal of dead cells
Removal of cell debris, inhibitory by products
High medium use, costs raw materials and sterilization.
IMMOBILIZED CELL SYSTEMS
High cell concentrations
Cell reuse
Eliminates cell washout at high dilution rates
High volumetric productivities
May provide favorable microenvironment
Genetic stability
Protection from shear damage
Mass transfer (diffusional) resistance
MAJOR LIMITATION
Whole cells provide cofactors, reducing power, energy that many enzymatic reactions require.
ADVANTAGE OVER
IMMOBILIZED ENZYMES
_____________: similar to enzyme immbolization. Entrapment and binding.
Active Immobilization
_____________: Biofil-multilater growth on solid surfaces.
Passive Immobilization
PHYSICAL ENTRAPMENT
Widely used method of cell immobilization.
Various matrices: porous polymers (agar, alginate, carrageenan, polyacrylamide. Chitosan, gelatin, collagen)
Porous metal screens
Polyurethane
Silica gel
Polystyrene
Cellulose triacetate
Polymer beads are also typically used
Encapsulation
Macroscopic membrane-based reactors (hallow fiber)
Method of preparing polymer beads:
Gelation of polymers
Precipitation of polymers
Ion exchange gelation
Polycondensation
Polymerization
There is direct contact between nutrient and suppor materials.
PHYSICAL ADSORPTION
High cell loadings
PHYSICAL ADSORPTION
Disadvantages: porous support materials causes intraparticle pore diffusion (at high cell densities) and hard to control microenvironmental conditions
PHYSICAL ADSORPTION
Selective of suitable support materials is highly based on adsorption capacity and strength of binding
PHYSICAL ADSORPTION
In general, good support materials should be rigid and chemically inert, should bind cells firmly, high loading capacity.
COVALENT BINDING
Widely used for enzymes but not for cells.
COVALENT BINDING
Multilayer growth of cells on solid support surfaces
PASSIVE IMMOBILIZATION:
BIOLOGICAL FILMS
Support materials can be biologically active or inert
PASSIVE IMMOBILIZATION:
BIOLOGICAL FILMS
Common in waste water treatment and mold fermentations
PASSIVE IMMOBILIZATION:
BIOLOGICAL FILMS
DIFFUSIONAL LIMITATION
Analysis similar to immobilized enzymes.
Damkoler number
Effectiveness factor
Thiele modulus.
___________: feed flows through a column packed with immobilized cells. Similar to a plug flow reactor. Can be recycles chamber.
Packed-column
_________: feed flows up through a bed of immobilized cells, fluidizing the immobilized cell particles.
Fluidized-bed
________: air bubbles suspend the immobilized cell particles in a reactor.
Airlift
SOLID-STATE FERMENTATIONS
Fermentation of solid materials
Low moisture levels or water activities
Agricultural products or foods
Smaller reactor volume
Low contamination due to low moisture
Easy product separation
Energy efficiency
Differentiate microbiological structures
Cellular metabolism produces heat, removed by internal coils or reactor jackets.
Heat Removal
Cellular metabolism produces compounds that promote foaming. Controlled by mechanical foam breakers and chemical additives.
Foam Control
Cellular respiration requires oxygen. Sparged air, impeller makes smaller bubbles and increases residence time.
Providing Oxygen
Single organism desired. Steam and filtering.
Sterilization
Good oxygen mass transfer
High energy requirement for mixing
Seal to maintain, keep sterile.
Agitated Tank
Low shea environment
No seal needed
Restricted to low viscosity
Less mixing than agitated tank
Bubble coalescence limits air flow rate
Bubble Column
Better mixing than bubble column with same low shear and energy requirements and lack of seal.
Work with higher viscosity liquids than bubble columns.
Still less mixing than agitated tank
Loop reactors
WHAT FACTORS LIMIT
SIZE OF REACTORS
Ability to provide oxygen and remove heat.
REACTOR TYPES
Stirred-tank
Bubble Column
Airlift
Propeller Loop
Jet Loop
Impeller breaks bubbles into smaller ones to provide for better oxygen mass transfer
AGITATED TANKS
are typically glass, commercial fermentors are typically stainless steel
Bench-top tanks
Heat removal/addition is typically by coils along the wall, or a water jacket around the tank
AGITATED TANKS
prevents foaming problems, but can cause additional mass transfer resistance
Antifoam
volume of liquid in tank; does not include head space
“working volume”
must not allow contamination
Seal for agitator shaft
are used to augment mixing and gas dispersion
Baffles
__________: disc with 6 to 8 blades. Pumps fluid in a radial direction. Compartmentalization with multiple impellers on a shaft.
Rushton impellers
_______________: pumps liquid in a vertical direction. Lower energy for the same oxygen mass transfer. Lower shear rates.
Axial flow impeller
OXYGEN MASS TRANSFER
Bulk gas phase oxygen concentration
Tranfer across stagnan gas layer
Partitioning into the liquid phase (C* at saturation)
Transfer actress stagnant liquid layer
Bulk liquid concentration (
CL)
Transfer across stagnan liquid layer to cell.
Transfer rate at _________ is determined by the slowest rate (just like on a highway)
steady state
______ is not the rate at which you provide air to the reactor. You will actually provide much more oxygen to the reactor than is transferred to the cells.
OTR
Utilizes a ________ with actively growing cells.
fermentor
The air to the fermentor is shut off, and the DO decreases due to consumption by the microorganisms. The air is then turned on, and the the DO increases.
DYNAMIC METHOD
Make the controlling regime the same on the small scale as on the large scale.
SCALE-UP
Empirical
SCALE-UP
SCALE-UP CRITERION
Power Input - OTR
Liquid circulation rate - mixing time
Tip speed - shear
Reynolds number - geometry
________ by requiring characteristic times to be constant between the small and large scale.
Scale-up
Many types of characteristic times are related to mixing, diffusion, oxygen mass transfer, reaction, and growth.
ANOTHER METHOD
COMMON ON-LINE INSTRUMENTATION
pH
Temperature
Dissolved oxygen
Foam
Flow Rates
Level
Off-gas composition (CO2,O2,VOCs)
is generally not as sophisticated as chemical production process control due to a lack of on-line sensors.
Fermentation process control
Each probe into the ____________ the probability of contamination, difficult to sterilize some probes, probe fouling, probe placement (gradients within the fermentor).
fermentorincreases
Form a group of three and describe 5 control loops based on the most common instrumentation.
TYPICAL FERMENTOR
CONTROL SCHEMES
Identify the measured variable, and the controlled variable-specifying what is the final control element (ie. valve, pump, etc.)
TYPICAL FERMENTOR
CONTROL SCHEMES
the absence of detectable, viable organisms.
Sterilization
reduction in the amount of detectable, viable organisms.
Disinfection
__________: some portion of the population is more resistant to sterilizing agents than other portions.
Sterilization is probabilistic
______: Heat-sensitive liquids and gases. Most common for gases - P important.
Filter
_______: Most common for liquids and equipment. Steam. Typically 121°C.Time and T are important. Risk degrading medium components.
Heat
_____: Surfaces.
Radiation
_____: Risk of toxic residues.
Chemical
______ - a faster growing contaminating organism can outgrow the desired organism and cause a washout of the desired organism.
Chemostat
_________ - the product can be biologically contaminated (could be lethal) or the purity profile could be significantly effected (indust. fermentations 100 kl).
Batch
to clean with the purpose of removing possible biological and nonbiological threats to human health.
Sanitize
to greatly reduce the number of living organisms.
Disinfect
to eliminate all viable organisms present (often our goal).
Sterilize
(filtration equipment, reactors, etc.) can be sterilized by heat, microfiltration, radiation, chemical agents, and UV light.
Fluids and process equipment
a cell, spore, or virus that is dead will not reproduce (cells and viruses) or germinate (spores) under conditions favorable for growth (opposite is “viable”)
Death
is a common method.
Thermal sterilization
is common for the insides of reactors that can’t be heat or steam sterilized.
Ethylene oxide
(heat labile vitamins, proteins, sugars) must be filter sterilized using filters with narrow pore-size distributions.
Media that can’t be heat sterilized
70% v/v ETOH in water with HCl to pH 2 is a ____________________.
good sterilizing fluid
is commonly used to sterilize filtration equipment.
Weak (3%) sodium hypochlorite solution
GENERAL APPROACH
1.Separation of insoluble products or Components.
2.Primary isolation or concentration and removal of water.
3.Purification and removal of contaminated chemicals.
4.Product preparation.
FACTORS THAT IMPACT DIFFICULTY
AND COST OF RECOVERY
1.Product can be biomass, intracellular, or extracellular components.
2.Fragile or heat sensitive.
3.Concentration or titer in the broth.
4.Typically, recovery and purification are more than 50% of total manufacturing costs.
INSOLUBLE PRODUCTS OR COMPONENTS
Filtration
Centrifugation
Coagulation and Flocculation
Most cost-effective, most common in industrial biotechnology.
Filtration
Rotary vacuum precoat filters: traditional. Penicillin mold.
Filtration
Cross-flow ultrafiltration: 0.02-0.2
um bacterial separations.
Filtration
Cross-flow microporous filtration 0.2-2
um for yeast.
Filtration
Used to separate solids of size. 01 um to 100 um using centrifugal forces.
Centrifugation
Being replaced by microfiltration
Centrifugation
Pretreatment to centrifugation, gravity settling, or filtration to improve separation.
Coagulation and Flocculation
________: formation of small flocs of cells using coagulating agents electrolytes.
Coagulation
________: formation of agglomeration of flocs into settleable particles using flocculating agents, polyelectrolytes, or
CaCl2
Flocculation
Used wastewater treatment processes to improve clarification.
Coagulation and Flocculation
Mechanical Methods
Sonication
Bead beating
Pressing
Non-Mechanical Methods
Osmotic shock
Freeze-thaw
Enzymatic
_______: disrupts cell membrane. Mostly used at the laboratory scale.
Ultrasound
_________: extrude cell paste at high pressure.
Pressing
__________: grind cells with glass, metal beads.
Bead beating
________ is a problem with all of these methods.
Heat dissipation
____________: salt differences to cause the membrane to rupture. Common.
Osmotic shock
________: Causes cell membrane to rupture. Common.
Freeze-thaw
_________: Lysozyme attacks the cell wall.
Enzymatic
SEPARATION OF SOLUBLE PRODUCTS
Liquid-liquid extraction
Aqueous two-phase extraction
Precipitation
Adsorption
Dialysis
Reverse osmosis
Ultrafiltration and microfiltration
Cross-flow filtration and microfiltration
Chromatography
Electrophoresis
Electrodialysis
Separate inhibitory fermentation products from broth.
LIQUID-LIQUID EXTRACTION
Based on the solubility difference for the compound between the phases.
LIQUID-LIQUID EXTRACTION
PRECIPITATION
Solubility reduction at low temperatures (less than - 5°C) by adding organic solvents.
- inorganic salts (NH4)2SO4 at high ionic strength.
Salting out
Membrane separation is used to remove low molecular weight solutes.
DIALYSIS
Used to remove salts from protein solutions.
DIALYSIS
Transport occurs due to a concentration gradient driving force.
DIALYSIS
Sal phase becomes more concentrated.
REVERSE OSMOSIS (RO)
pressure is applied to the salt phase, causing water to move against a concentration gradient.
REVERSE OSMOSIS (RO)
_________: Transport of water molecules from a high to a low concentration of pure water to salt water.
Osmosis
Pressure-driven molecular sieve to separate molecules of different sizes.
ULTRAFILTRATION AND MICROFILTRATION
____________: retained components accumulate on the filter. Gel layer formed on the filter.
Dead end filtration
_____________: retained components flow tangentially across the filter.
Cross filtration
Separates mixtures into components by passing the mixture through a bed of adsorbent particles.
CHROMATOGRAPHY
Solutes travel at different speeds through the column resulting in the separation of the solutes.
CHROMATOGRAPHY
Highly specific interaction between a ligand on the particle and a component in the mixture. Often based on antibodies.
AFFINITY CHROMATOGRAPHY
Separation of molecules based on size and charge in an electric field.
ELECTROPHORESIS
Membrane separation to separate charged molecules from a solution.
ELECTRODIALYSIS
FINISHING STEPS
Crystallization
Drying
