Week 10 - Biotechnology

Biotechnology

Use of biological processes in organism to produce goods, grouped into red, white, and green biotechnology

Sources of microbes

Scientists maintain culture collections of modified microbes that are often frozen or freeze-dried for preservation

Bioprospecting

Searching for new + useful microbes to cultivate

Industrial fermentation

The process of cultivating microbes to produce desired substances using bioreactors to maximize cell density + product yield

Fed-batch reactors

Type of bioreactor where feed (nutrients) is added untul the max cell concentration is reached, then biomass is harvested

Chemostat

Continuous cycling of feed and effluent (biomass), so that products are harvested indefinitely

Primary metabolite

Products of metabolism required for cell growth, production matches the cell growth curve

Secondary metabolite + what phase are they produced in

Products of metabolism not needed for cell growth, often produced in the stationary phase

Random mutagenesis + cons

Cell genomes are randomly mutated via chemical/radiation exposure, then screened for desired mutations. Difficult to do, can produce negative mutations, expensive, and little is known about mutation outcomes

Site-directed mutagenesis + protein example

Specific sites in DNA are mutated, a more rational approach than random mutagenesis. Ex. can stabilize proteins by adding disulphide bonds, add more cysteine AAs

Oligonucleotide-mediated site-directed

Complementary oligonucleotide containing desired sequence change anneals to ssDNA, turning it into dsDNA plasmid. Transformed in E. coli to produce desired plasmid

PCR site-directed

Complementary primers with the desired mutation undergo PCR → Dpnl restriction enzymes cleave the original template DNA → recover the desired mutated product in E. coli

Directed enzyme evolution

Same as random mutagenesis but involves 1 gene instead of the whole genome. Build a variant library of the gene, then screen library for the best genes and mutate them

Methods to build a variant library for directed enzyme evolution (2)

• Error-prone PCR: PCR with a bunch of mistakes so you generate variation

• DNA shuffling: shear up mutated bits of DNA + stick them randomly back together (“chimeras”)

Frances Arnold

Won the 2018 Nobel prize for inventing directed enzyme evolution

Describe the bacterial mechanism that CRISPR-Cas genome editing is based on

Bacteria exposed to pathogens will insert a copy of their DNA into repeat segments + transcribe it into crDNA. crDNA binds to Cas proteins to form CRISPR-Cas surveillance complexes that police the cell for the pathogen. If they find the pathogen, they bind to it and target it for destruction

CRISPR locus

Consists of unique spacer segments containing phage genomes that were previously encountered

Cas proteins

Process pre-crDNA transcripts into mature crDNA, then bind to it to form CRISPR-Cas surveillance complexes

Cas 9

A Cas protein that breaks down DNA based on crDNA instructions, can be altered + used to edit genomes by replacing sequences

Production of recombinant proteins

Inserting DNA sequences of interest into plasmids using expression vectors and fusion proteins

Expression vectors + difficulties

Encode for the protein of interest. Contain promoter, operator, Shine-Dalgarno sequence, and protein of interest. Difficult because they require no introns, proper glycosylation, and disulfide bond formation

Fusion proteins

Combination of elements from multiple proteins, which are purified using affinity chromatography (tagged fusion proteins stick to the column while others pass through).

Synthetic biology

Designing biological systems to carry out tasks, artificial genome is put into a surrogate cell + replaces the OG genome

BioBricks

Used for vector assembly, contains restriction sites that allow parts to be removed + inserted

Red biotechnology + 2 main goals

Microbes for medical applications, goal is to:

1. Produce secondary metabolites with therapeutic properties

2. Host the production of recombinant human proteins

Secondary metabolites used in red biotech

Antibodies, statins, botox

Insulin

The first human recombinant protein to be made + marketed

Antibiotics

Inhibit bacterial growth by interfering with the 70S ribosome, cell wall synthesis, membrane integrity, DNA synthesis, and folic acid synthesis

Mechanism of penicillin + yield in red biotech

B-lactam antibiotic, inhibits the formation of peptidoglycan cross-links by binding to DD-transpeptidase. Yield was refined via mutagenesis

White biotechnology

Industrial applications, using microbes to convert cheap biomass into high value products

Biorefinery

Converting biomass into products

Cellulose/hemicellulose

Fraction of plant biomass used as feedstock for biofuel production

Biofuels + types

Biomass → fuel, includes ethanol (product of yeast fermentation) and butanol/acetate (product of C. acetobutylicum fermentation, better than ethanol)

Bioplastics + types

Polyhydroxybutyrate (PHB) and polyhydrozyalkanoate (PHA) are biodegradable polyesters produced by microbes

Industrial enzymes

Facilitate many commercial processes such as high-fructose corn syrup production and laundry detergent, can be improved via bioprospecting and mutagenesis

Vitamins and amino acids + white biotech

Can be produced by microbes, which are handy since they are stereospecific and only produce L isomers

B vitamins

Very complicated, produced by Pseudomonas + Propionibacterium

Overproduction of lysine in cells

Caused by genetic manipulation that removes feedback inhibition to boost production

Green biotechnology

Genetically modifying crops to improve nutrition and yield

Agrobacterium + what is it a cool example of

Use to introduce modified genes into plants via A. tumefaciens. Example of cross-kingdom DNA transfer

Mechanism of A. tumefaciens in green biotech

Induces crown gall tumours in plants by inserting tumour-producing plasmids into plant cells, causing the production of phytohormones and opines. Can replace phytohormone and opine genes with desired genes in T-DNA to introduce them into plants

Protoplast formation

Removing the cell wall of plants in order to introduce new DNA

Biolistics

Shooting metal fragments coated in DNA into plant cells

Herbicide resistance

Make resistant transgenic plants by introducing EPSP enzyme using biolistics, making them resistant to Roundup

Insect resistence

Makes resistant transgenic plants by encoding cry genes on plasmids to produce Bt toxins, which kill insects

Biofortification + example

Engineering higher nutrient content in crops, ex. more vitamin A in golden rice combats nutritional deficiencies in third world countries

Intrinsic vs extrinsic factors of food spoilage

Intrinsic is of the food (ex. water content, nutrient content, pH + buffers, structure), extrinsic is of the environment (ex. temp, humidity, gases)

Desirable food spoilage of milk

Raw milk is processed with microbes, making it less favourable for the growth of spoilage microorganisms

Ways to minimize food spoilage (6)

1. Reduce water activity of food: dry it, add solutes

2. Control temp

3. Increase acidity: pickling

4. Chemical preservatives: artifically via sodium benzoate and nitrites, naturally via bacteriocins, lactic acid, and acetic acid

5. Irradiation

6. Modified-atmosphere packaging (MAP): vacuum seal to take away O2 or flood package with CO2 (or give red meat O2)

Fermenting food with mold

Used in Asia (ex. miso, soy sauce, sake). Koji and maromi are cultures of Aspergillus mold, bacteria, and yeast used to ferment grains

Making vinegar with acetic acid bacteria

Convert ethanol → acetic acid, use trickle/quick method to pass ethanol though a bed of bacteria, supply O2, and collect acetic acid at the bottom

Foodborne intoxication vs infection

Presence of toxins (quick onset of symptoms) vs microbes (delay in symptoms)

Domestic contaminants of water

Includes suspended solids, organics, pathogens, and parasites

Persistent organic pollutants (POPs)

Pollutants, heavy metals, inorganics

Wastewater treatment plant + goals

Discharge cleaned effluent into water bodies, a continuous culture process. Reduce total organic content, remove harmful pathogens, remove POPs, and remove inorganic compounds (like NH4, N2, P),

Process of treating wastewater

1. Pre-treatment: physically remove large objects

2. Primary treatment: physically remove primary sludge (sediments and grease)

3. Secondary treatment: use trickling filter + activated sludge unit to break down organics using biofilms

4. Tertiary treatment: filtration

5. DisinfectionL chlorination, UV light exposure, or ozonation

Trickling filter

Used in secondary treatment. Wastewater is sprayed on top of biofilm growing on a bed matrix, which removes pollutants via metabolism

Activated sludge systems

Used in secondary treatment. Flocs (clumps of microbes and absorbed material) form, which are removed as they settle

Anaerobic sludge digester

Last step in secondary treatment, digests organic waste while biosolids (indigestible) get incinerated or used as fertilizer

Drinking water purification

Same as wastewater treatment but cannot contain any indicator organisms (cause disease)

Walkerton. Ontario

Suffered a giant E. coli outbreak due to contamination of drinking water