Module 8: Microbial Biotechnology (Basic Concepts + Techniques) Flashcards
Biotechnology
The use of biological processes, systems, or organisms for the production of goods or services (intended to improve quality of human life)
What made the growth of biotech possible?
Molecular biology tools
–> And therefore, the study of microbial genetics (as this study led to the development of these tools)
What are the major categories of biotech?
Red = Biotech for medical use
White = Biotech for industrial use
Green = Biotech for agricultural use (NOT environmental SARAH)
What is the extra “4th” category of biotech applications?
Biotech for use in the environment (bioremediation)
The microbes utilized in biotech are often _______________ versions of __________________ strains
Microbes in biotech are usually MODIFIED versions of naturally occurring microbes
Why do we modify microbes for biotech uses? (generally speaking)
To optimize them for some specific biotech function or condition that they are not fully adapted for
What are the main targets for microbial modifications in biotech?
1) A microbe’s ability to grow + replicate well under standard lab conditions
2) Increased production of some desired product (or process)
What are the main limitation of natural microbes in biotech?
(Essentially, why would they need to be modified?)
1) Are not adapted to grow well or replicate in a non-natural environment (lab)
2) Do not produce large amounts of a desired product
What are the 2 overarching ways in which we can get optimal microbes for biotech use?
1) We can FIND them –> selectively isolate the microbes exhibiting most optimal features
2) We can MAKE them –> use recombinant DNA technology to construct more useful microbes
How has the use of microbes increased efficiency of developing biotech?
1) Microbes have shorter generation times
2) Microbes can be handled + modified easily
(selection of microbes with superior qualities can be done in YEARS rather than the DECADES it would take for animals and crop plants)
Many useful microbes exist BUT before we can use them we must…
Know about them
–> We have to find them first!
What are the two main methods for finding microbes for some biotech purpose?
1) Using culture collections
2) Bioprospecting
Culture Collection
An archive of microbial strains for use in microbiology study and biotech
–> collections consists of PRESERVED living samples of microbial cultures
How are culture collections made?
Most culture collections are open source == scientists from all over deposit microbes they have isolated and characterized into these collections
How can scientists use culture collections to find microbes for a specific purpose?
By SCREENING the collections for those that exhibit properties or products of interest
What is a limitation of searching for microbes using culture collections?
In many cases, well suited microbes with desired properties may not have been found yet == will not be in the culture collection!
If a given microbe of interest cannot be found in a culture collection, what is the next step?
BIOPROSPECTING
Bioprospecting
Searching for novel organisms, biological materials, or processes in NATURE that can be used in biotech
–> uses a variety of search tools to find novel microbes from different environments and then screen them for specific activities
What is a limitation of bioprospecting?
(And what is providing a potential solution to this?)
Limitation = Low success rate at times due to many microbes not being able to be cultivated!
Potential solution = metagenomics (allows us to analyze uncultivated microbes)
Fermentation
Has a DUAL MEANING:
Biochemical Fermentation –> Specific catabolic reactions that produce ATP in the ABSENCE OF OXYGEN (anaerobic)
Industrial Fermentation –> Any industrial process involving the culture of microorganisms for the production of desired substances (can be aerobic OR anaerobic
Industrial fermentation and biochemical fermentation…
ARE NOT EQUIVALENT!
–> Industrial fermentation may USE biochemical fermentation but it does not have to!!!
Many modern fermentations occur in the presence of ____________ and no ____________ is occurring
Many modern fermentations occur in the presence of OXYGEN and no biochemical fermentation is occurring
–> STILL called fermentation!
Large scale production of recombinant proteins in E. coli in the presence of oxygen is an example of what?
FERMENTATION (in biotech!)
Bioreactors
Large culture vessels designed to maximize cell density and product yield during fermentation
(Where industrial fermentation takes place!)
What are bioreactors AKA?
Fermenters
Bioreactors are specially designed to precisely control _______________________ to _________________ production
Bioreactors are specially designed to precisely control environmental conditions to optimize production
What are the main conditions being controlled in bioreactors?
1) pH
2) Temperature
3) Oxygen content
4) Nutrient content + abundance
Bioreactors can be what kinds of systems?
OPEN or CLOSED systems!
Closed vs Open Bioreactor systems
Closed system = NO new nutrients are added or medium removed
(no interaction of culture with the exterior environment)
Open system = Nutrients are added to the medium and/or medium is removed from culture
(reactor contents have at least SOME interaction with the external environment)
Are closed or open bioreactors used more? (why?)
OPEN systems
== Provide greater opportunity for control! (and therefore greater optimization is possible!)
What are the two types of open bioreactors?
1) Chemostat
2) Fed-Batch Reactor
What is a type of closed bioreactor?
+ How does it work?
Batch Reactor
–> Everything for fermentation is added into a reactor and the process continues without ANY modifications; collection occurs at the end!
== Process goes to completion! (all substrate used and/or all cells die)
What are the features of a chemostat + how does it work?
Culture contents within the reactor maintain a CONSTANT volume
–> Due to dual input + output occurring at the same rate!
Input = adding new medium
Output = removing some of the culture mix (can contain products, cells, nutrients, etc)
Within chemostats a __________________ is maintained
A physiological steady state is maintained!
What is the purpose of a chemostat?
To maintain a constant growth rate within the culture
Why are chemostats continuous systems?
Because nutrients are continuously being added to the culture (constant input)
Within a chemostat how can growth rate/cell density be controlled?
1) Manipulating NUTRIENT COMPOSITION of the medium
2) Manipulating the FLOW RATE of fresh medium into the culture vessel
altering nutrient availability by these methods can increase or decrease growth rate
What is a benefit of the constant efflux within chemostats?
Allows for the removal of any potentially toxic or inhibitory byproducts that may form
What are the features of a fed-batch reactor + how does it work?
Culture contents within the reactor have a VARIABLE volume
–> Due to SINGLE input (NO OUTPUT)
Input = Intermittently adding new medium (input turned “on” + “off”)
Output = NO OUTPUT; all culture is harvested at the END
Why is a fed-batch reactor considered “partially-open” and “non-continuous?
Partially-Open = b/c it has input but no output so it’s not fully open
Non-continuous = b/c input occurs intermittently, input is NOT occurring constantly
What is the difference in volumes between chemostat + fed-batch reactors?
Chemostat = CONSTANT volume
Fed-Batch = VARIABLE volume
Fed-batch reactors maintain a HIGH___________________
High cell density!
How are toxic/inhibitory byproducts handled in fed-batch reactor?
The intermittent addition of limiting nutrients prevents the system from being able to enter overproduction state (when byproducts get made the most)
Growth-Limiting Nutrient
A nutrient NEEDED for growth = limits growth when NOT present!
Why are growth-limiting nutrients used as input in bioreactors?
To be able to control the growth rate (will only grow as much as the nutrient is provided by a researcher!)
Fed-Batch Reactor (Definition)
A partially-open system in which a growth-limiting nutrient is added OVER TIME o control growth rate resulting in high cell densities
How is growth rate and cell density controlled in fed-batch reactor?
WHEN + HOW MUCH limiting nutrient is added to the culture
What factors determine what bioreactor should be used?
1) Type of microbe being used
2) Nature of the desired end product
Which bioreactor is most commonly used? WHY?
The Fed-Batch Reactor is more commonly used due to:
1) Greater reliability
2) Greater reproducibility
Microbes in a CLOSED system exhibit a ………..
This consists of:
specific growth curve!!!
== 3 phases!
Consists of:
1) LAG phase
2) EXPONENTIAL phase
3) STATIONARY phase
What is each phase of a growth curve in a closed system? (define each)
LAG phase = low cell density, almost no cell growth/very very low growth rate
EXPONENTIAL phase = growth!
STATIONARY phase = almost no cell growth/ very very low growth rate BUT high cell density
(Death phase)
What growth phases correspond to what kinds of metabolites?
Exponential Phase = Primary metabolites produced
Stationary Phase = Secondary metabolites produced
Primary Metabolite
A product of metabolic process/es that are required for cell growth
(== only made when cells are growing! –> Exp. phase )
Secondary Metabolite
A product of metabolic process/es that are NOT required for cell growth
(== only made when cells are NOT growing –> stat. phase)
How can we “force” a culture to stay in a specific growth phase?
Why would we want to do this?
Growth phases can be maintained by controlling environmental conditions
We want to maintain a specific growth phase to optimize yield of a desired metabolite (primary or secondary)
Antibiotics vs Ethanol: Each are examples of what?
Antibiotics = Secondary metabolite
Ethanol = Primary metabolite
Why are primary metabolites difficult to achieve a high concentration of in culture?
Two potential reasons:
1) Because primary metabolites tend to turn their own pathways off!
–> Tend to be a regulator in negative feedback loop!
== decreases yield of product
2) Because primary metabolite is toxic in high concentrations (leading to death)
What are potential genetic alterations we can make to improve microbes for biotech purposes (4)?
1) Downregulate competing pathways
2) Downregulate pathways that lead to byproduct formation
3) Increase expression of genes encoding for enzymes of a given pathway (increases yield!)
4) Alter genes to make strain grow on a cheaper carbon source (decreases costs of production!)
What are the two main ways (methods) we can improve microbial performance in biotech?
1) Mutagenesis (2 types)
2) Recombinant Proteins
Mutagenesis
The process by which an organism’s genetic sequence is changed (the process of mutation)
What are the two types of mutagenesis?
Random mutagenesis
+
Site-Directed Mutagenesis
Random Mutagenesis
The process of introducing RANDOM mutations into DNA sequences typically by exposure to a mutagen
What mutagens can be used in random mutagenesis?
1) UV Light
2) X-Rays
3) DNA damaging chemical agents
After conducting random mutagenesis, what do we do to find microbes of interest?
Screen all the mutants for a given function or feature
What are the drawbacks of random mutagenesis? (4)
1) Mutations produced are largely undefined (little info on them)
2) Can produce detrimental or non-useful mutations
3) Effective screens are sometimes not available for a desired function/phenotype (= wouldn’t be able to isolate mutant of interest)
4) IF screen does exist –> The screening process is labor intensive, costly, and time consuming
What are the benefits of random mutagenesis?
1) Does not require known potential DNA targets in advance (= good for strains that we haven’t fully characterized yet!)
2) Allows for discovery of novel DNA targets/mutations!
What is the general process of strain improvement by random mutagenesis?
A process of multiple rounds of mutagenesis followed by screening for superior phenotypic characteristics
1) Mutate a strain
2) Screen for a desired feature
3) Take the BEST COLONIES
4) MUTATE these “best colonies”
5) Screen for the desired feature
6) Take the BEST COLONIES again
7) REPEAT
In what microbe was Penicillin originally discovered?
Penicillium nonatum
After initial discovery of penicillin, what was done?
What did this result in?
1) Bioprospecting == Found Penicillium chrysogenum produces MORE penicillin than P. nonatum
2) Random mutagenesis of P. chrysogenum
3) Screening == found strain with mutation that had even greater penicillin production!
Penicillium nonatum vs Penicillium chrysogenum
BOTH produce penicillin
–> P. nonatum = first discovered BUT produces less penicillin
–> P. chrysogenium = discovered later BUT produces MORE penicillin
Site-Directed Mutagenesis
A method that induces SPECIFIC mutations within a DNA sequence
–> the formation of KNOWN + SPECIFIC mutations at SPECIFIC/KNOWN DNA sites!
Site directed mutagenesis produces __________ which can be used to ______________ which random mutagenesis cannot!
Site directed mutagenesis produces precise genetic changes which can be used to change a specific AA in a protein which random mutagenesis cannot!
What are the drawbacks of site-directed mutagenesis?
1) Requires prior knowledge (we need to know what the sequence of a target site is)
–> Limits applicability can’t be used for microorgs that are poorly characterized
What are the main methods of site directed mutagenesis?
1) Oligonucleotide Directed Mutagenesis
2) PCR Directed Mutagenesis
Oligonucleotide Directed Mutagenesis
A method for SD-mutagenesis that utilizes an oligonucleotide (primer) that is MOSTLY homologous to the target DNA sequence BUT contains a desired mutation
What is the process for Oligonucleotide Directed Mutagenesis?
1) Source DNA inserted into a vector
2) Recombinant vector is rendered single stranded
3) Designed primer w/ mutation is added to the mix + anneals (mostly) to the target site on the inserted DNA of the plasmid
4) DNA polymerase begins DNA synthesis of second strand starting from the primer end!
= dsDNA vector with mutation! (looks like a bubble in the molecule)
5) Vector is transformed into host cell
6) Host cell replicates + vector is divided into two ssDNA template strands for replication
7) Daughter cells produced have different genes! –> One has mutated variant + one has regular variant
8) SCREEN (for cells with the mutation)
How is the oligonucleotide with mutation able to hybridize to the DNA of interest if there is a mismatch?
Due to there being a large proportion of the primer that IS complementary == overrides the negative interaction of the mismatch!
What is special about the oligonucleotide in SD mutagenesis?
It is NOT completely complementary to the DNA of interest! (has desired mutation in it!)
What vector is commonly used for oligonucleotide directed mutagenesis?
M13 == Phage vector –> b/c it already has an ssDNA genome!
PCR Directed Mutagenesis
A method of SD mutagenesis using PCR (to produce fully mutated vector!)
What restriction sites are used in PCR directed mutagenesis?
DpnI
What is special about DpnI sites?
Will only get cleaved by restriction enzyme if DpnI sites are METHYLATED
What vector is usually used for PCR directed mutagenesis?
Usually a vector with 2 methylated DpnI restriction sites!
Process of PCR Directed mutagenesis
1) DNA of interest inserted into vector between two methylated DpnI sites
2) Recombinant vector is denatured = 2 single stranded vectors
3) Primer with desired mutation is added to the mix + anneals to the target DNA
4) DNA polymerase is added + a few rounds of PCR are conducted to produce different recombination products
== One of which is a molecule with TWO mutated strands! = fully mutated + NO methylated DpnI!
5) All products are transformed into E. coli
6) All NON fully mutated PCR products are cleaved at methylated DpnI site! –> Leaves only the mutated products!
In PCR directed mutagenesis, what is the role of DpnI?
Degrades any transformed molecules made from a NON-mutated strand (and as such HAS the methylated DpnI)
== Makes it so that only mutated copies persist!
In PCR directed mutagenesis, why is the DpnI cleavage done INSIDE E.coli?
Allows for DNA outside the cell to continue doing PCR and stuff (as no RE is outside cell to cleave the DNA) == will produce more mutated copies overtime!
What is an issue of SD-mutagenesis that recombinant protein method attempts to address?
SD-mutagenesis only amplifies and introduces mutated DNA into a given cell BUT there is no guarantee it will get expressed and lead to a product!
Recombinant protein method ensures that the mutated DNA WILL get expressed!
What are expression vectors?
Agents (usually plasmids) that are specifically designed to produce recombinant proteins within a host cell
Recombinant Proteins
Proteins produced by cells engineered with a specific gene (protein made from recombinant DNA)
Expression vectors are beneficial because they…
They increase the production of a desired protein!
What is the most common use of expression vectors?
Production of therapeutic human proteins
Examples of recombinant proteins produced using expression vectors (4)
1) Insulin
2) Growth Hormone
3) IL-2 (Interleukin-2)
4) Antiviral Interferons
Expression of a eukaryal gene in a bacterial cell will only occur if…
If the eukaryal foreign gene contains the correct bacterial transcriptional and translational elements
What are necessary elements for transcription + translation in bacteria? (6)
For Transcription:
1) Promoter (bacterial specific)
2) Operator
3) Terminator Sequence
For Translation:
1) Shine-Dalgarno Sequence
2) Start codon ~6-10 BPs away from the SD sequence
3) Stop codon
What does an expression vector contain for a eukaryal gene? (8)
1) Shine-Dalgarno Sequence (SD) –> 6-10 BPs upstream start codon
2) bacterial promoter + operator
3) cDNA version of eukaryal gene positioned 6-10 BPs from SD sequence
4) MCS in the eukaryal gene RIGHT after start codon
5) Terminator sequence
6) Origin of replication for bacterial host cell
7) Selectable marker
What is unique about the promoter in an expression vector?
VERY strong promoter!
It is a customized promoter designed to increase level of transcription
What is the optimal positioning of a gene’s start codon relative to the shine-dalgarno sequence?
Start codon should be 6-10 BPs downstream of the SD sequence
What is different about a eukaryal gene of interest that must get inserted into an expression vector?
WHY?
The gene must be in cDNA format!
Bacteria DO NOT have introns! As such, all the non-coding portions of a eukaryal gene must be removed before inserting into expression vector
What is the purpose of the terminator sequence in an expression vector?
Signals for the end of transcription
(Sequence must be bacteria specific!)
What is the problem with eukaryal EV recombinant proteins?
May not end up being functional within the bacterial host!
Due to differences in:
1) glycosylation + 2) Disulfide bond formation (improper formation)
between bacterial + eukaryal systems
What is different between bacterial and eukaryal protein glycosylation?
They typically glycosylate with different sugars!
–> Bacteria = Mannose Units
–> Eukarya = Sialic Acid
What is different for EV vectors being introduced into a eukaryal host?
1) No SD sequence
2) Terminator sequence should signal for addition of Poly A tail
What are fusion proteins?
A form of recombinant protein that contains the domains of 2+ proteins
What are fusion proteins AKA?
TAGGED PROTEINS
What are fused proteins made of usually?
1) Protein of interest
2) Portion of another protein with some beneficial characteristic (LIKE AN AFFINITY TAG)
What is an Affinity Tag?
A peptide sequence that facilitates PURIFICATION of a recombinant protein (allows for its isolation from other cellular materials)
What is affinity chromatography?
A method of using chromatography to separate out proteins with affinity tags from a sample
Process of Affinity Chromatography of a Recombinant Protein
1) EV created with gene of interest fused to affinity tag DNA sequence
2) EV inserted into host cell
3) Transcription + protein sythesis of the tagged protein
4) Lyse cells + collect resulting lysate (releases proteins)
5) Pass the lysate through a chromatography column lined with beads that have a receptor adhered to them
== Affinity tags of the recombinant proteins bind to these receptors + immobilize the proteins of interest in the column
6) WASH the column = removes any other non-bound materials from the column
7) Tag removal
= Isolated protein of interest
Synthetic Biology
The CONSTRUCTION of NOVEL biological systems and functions from constituent parts
–> the design and building of operating systems with defined specifications to carry out desired tasks
Oligonucleotide Synthesizers
Computer-controlled instruments that automatically synthesize oligonucleotides
Synthesizing truly synthetic organisms would require an entire microbial genome to be… (4)
1) Synthesized
2) Introduced into a cell
3) Replicate in the cell
4) REPLACE preexisting host cell DNA
Xenobiology
A developing field focusing on the development of novel biological systems through expansion of the genetic code and incorporation of novel AAs into proteins
–> A subfield of synthetic biology that studies and designs biological systems using NON-NATURAL biochemical building blocks + genetic codes
What are 2 examples of successes in xenobiology?
1) E. coli successfully altered so that its STOP codon actually encodes for phosphoserine (a novel AA)
2) E. coli with an A-T base pair replaced by an entirely synthetic nucleotide pair
“Biological Parts”
(plus examples)
“Standard” genetic elements
Ex:
1) Enzyme encoding genes
2) Regulatory DNA sequences
3) Genes encoding regulatory proteins
General proposed process for the construction of a synthetic organism
1) Computer used to design a genome that will produce an organism with desired properties
2) Produce the synthetic genome
3) Insert synthetic genome into surrogate cell
4) Synthetic genome replaces host cell genome
== Synthetic organism with desired properties
What are biological parts analogous to?
Standard parts in engineering (nuts and bolts)
Registry of Standard Biological Parts
Contains descriptions of these biological parts (ex: promoters, ORFs, ribosome binding sites, etc.)
–> Characterizes a set of “pieces” that could be put together to make a desired organism
What does a Biobrick vector consist of?
Vector with a biological part flanked by two pairs of restriction sites
Left side of part = EcoRI + XbaI
Right side of part = SpeI + PstI
(and a selectable marker)
What is the biobrick process?
1) One of the biobrick vectors is cleaved with EcoRI + XbaI == OPENED VECTOR (will become the backbone of the “assembled part”)
2) A second biobrick vector is cleaved with EcoRI + SpeI = CUT OUT BIOBRICK
3) The two fragments containing the two biobrick parts are annealed
–> EcoRI ends come together
–> XbaI + SpeI ends come together
4) = Assembled part –> New biobrick vector with the 4 restriction sites and TWO joined biological parts!
XbaI + SpeI
Two restriction sites whose ends can anneal to one another (as 4 out of 6 bases are complementary!)
–> Ligating these ends will “destroy” the restriction sites!
