Biotech Exam 3

Microbes (microorganisms)

tiny organisms that are too small to be seen individually by the naked eye

(Bacteria, fungi, algae, and protozoa)

What percentage of all bacterial species have been identified, cultured, studied, and estimated to compromise over 50% of the earth”s biomass?

less than 1%

Bioprospecting

the discovery and development of new products from biological resources

Microbial enzymes

used in applications from food production to molecular biology research

Examples of microbial enzymes

Taq DNA polymerase, cellulase, subtilisn

Taq polymerase as a tool:

heat stabile, isolated from a thermophile

Cellulase as a tool:

- makes animal food more easily digestible

- stone-washed jeans

Subtilisin as a tool

laundry detergent

The Yeast Two-Hybrid System

determine if proteins ‘A’ and ‘B’ interact with each other where two fusion proteins are created

One fusion protein of Yeast Two-Hybrid System

fusion of protein ‘A’; the “Bait” with a DNA binding domain (DBD) of a transcription factor that binds to the promoter.

Second fusion of Yeast Two-Hybrid System

fusion of protein ‘B’; the “Fish” with an activator domain (AD) that binds to the enhancer

What happens when protein ‘A’ and ‘B’ interact?

the DNA binding domain and the activator domain will be brought together where this active transcription initiation complex results in expression of a specific reporter gene, such as lacZ

The main purpose of the Yeast Two-Hybrid System is to test for the interaction of different ______ molecules.

protein

What is CRISPR?

clustered regularly interspaced short palindromic repeats, the repeats are palindromic, and each repetition is followed by a short segment of spacer DNA

Prokaryotic immune system that provides acquired immunity and resistance to pathogens

CRISPR

What percentage of CRISPR is found in sequenced bacterial genomes?

approximately 50%

Spacer DNA in CRISPR:

is from previous exposures to foreign DNA (ex: virus or plasmid)

Where are Cas genes located?

next to CRISPR sequences

What does Cas9 must be complexed with to cleave a target DNA?

complexed with a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA)

What is used for genome editing

CRISPR/Cas9-gRNA complex

When was CRISPR first recognized?

1987 by scientist Yoshizumi Ishino

How are microbes used for everyday applications?

food products, fermentation, therapeutic proteins

How are microbes used in food products?

used with traditional and modern biotechnology including bread, yogurt, cheese, and alcoholic beverages

When was the first recombinant DNA food ingredient approved by the Food an Drug Administration (FDA)?

1999, it was a recombinant form of an enzyme to make cheeses

What is extracted from calves’ stomachs?

enzyme rennin which is used for curds to make cheese

What is chymosin?

a rennin that was cloned and expressed in bacteria, it is less expensive and easier to produce

Fermentation

process of deriving energy from sugars in the absence of oxygen

Lactic acid fermentation is used to make:

yogurt, sour cream, sauerkraut, vinegar and certain cheese breads

Alcohol fermentation is used to make

beer, wine, champagne

Therapeutic proteins

bacteria are used to produce medically important proteins

What was the first recombinant molecule expressed in bacteria for use in humans?

insulin, was genetically engineered in 1978, commercially available in 1982

Who developed the first vaccine? When did it happen?

Edward Jenner in 1796

What was Edward Jenner’s vaccine for?

Dr. Jenner used live cowpox virus to vaccinate James Phipps against smallpox.

Antigen

foreign substance that stimulates an immune response

What are antigen?

-whole bacteria, fungi, and viruses

-proteins, lipids, or carbohydrates

Immune system responds to antigens by producing antibodies called _____

antibody-mediated immunity

What cells recognize and bind to the foreign antigen?

B cells

With the help of T helper cells, what do B cells do?

they undergo activation to form plasma cells that produce antibodies

Antibodies bind to ______

foreign antigen

Once antibodies bind to the foreign antigen, what does macrophage do?

it can recognize the antigens coated with antibodies and phagocytize them

Can our natural production of antibodies protect us from all pathogens?

no, sometimes it is not enough

What are vaccines?

it’s a part of a pathogen or whole organisms that can be given to humans or animals by mouth or by injection

What do vaccines stimulate?

they stimulate the immune system against infection by those pathogens

Four major strategies to make vaccines

-subunit vaccines (most important)

-attenuated vaccines

-inactivated (killed) vaccines

-mRNA vaccines

How are subunit vaccines made?

by injecting portions of viral or bacterial structures (ex: purified proteins)

What do attenuated vaccines use?

uses live bacteria or viruses that have been weakened through aging or by altering their growth conditions to prevent replication

How are inactivated (killed) vaccines made?

by killing the pathogen and using the dead or inactivated microorganism for the vaccine

What are mRNA vaccines?

non-replicating mRNA encased in a lipid nanoparticle coat so it can enter cells (ex: Pfizer/BioNtech and Moderna vaccines)

How are majority of subunit vaccines made currently?

using recombinant DNA approaches in which the vaccine is produced in microbes

Hepatitis B

-genes for proteins on outer surface of virus are cloned into plasmids and transformed into yeast

-fusion proteins produced by the yeast are purified

What vaccine protects us against the nine strains of human papillomavirus (HPV)

Gardasil

How many targets for vaccine development are biotechnology companies working on?

50 targets

What are some of the targets for vaccine development?

-Influenza

-Tuberculosis

-Malaria

-HIV

-Coronavirus (Covid-19)

Why are microbial genomes sequenced?

to identify genes involved in bacterial metabolism, cell division, and genes that cause human and animal illnesses

What is another reason why microbial genomes are sequenced?

to find new strains:

-for bioremediation or other tasks

-identify disease causing organisms

What does Streptococcus pneumoniae cause?

causes ear and lung infections, killing 3 million children worldwide each year

The Streptococcus pneumoniae vaccine:

had low efficacy in children

In 2001, the Streptococcus pneumoniae genome was sequenced and _____

many genes encoding proteins on the surface of the bacteria were discovered, which led to new vaccines and treatments

What is metagenomics?

the study of genetic material recovered directly from environmental samples

What collective genomes of microorganisms inhabit a particular environment, especially on/in the human body?

microbiome

What plans did the NIH announce in 2008?

The Human Microbiome Project

Human Microbiome Project

a five year project to sequence 600 genomes of microorganisms that live on and inside humans, currently over 5,000 genomes catalogued

What are the goals of the The Human Microbiome Project?

-determine if individuals share a core human microbiome

-understand how we acquire and maintain microbial communities

-understand how changes in the microbiome can be correlated with changes in health and conditions that affect the microbiome

-develop new methods for analysis of the microbiome

-address ethical, legal and social implications raised by human microbiome research

What is a technique used to detect and track microbes?

Microbial diagnostics

Bacterial Detection Strategies

RFLP analysis (done with southern blott) , PCR, and DNA sequencing

Data bases are available for comparison of clinical samples

used to detect and track bacterial contamination of food

What are microarrays used for?

to track contagious diseases

What new approaches have microarrays created?

new approaches for detecting and identifying pathogens and for examining host responses to infectious diseases (50,000 species tested in parallel)

what are microarrays also used for?

to find “signature” changes in gene expression for a particular pathogen

How much arable land has increased despite the world population nearly doubling in the past 40 years?

10%

What has improved crop breeding through traditional methods enabled?

plant geneticist to feed larger numbers of people

New modern methods have resulted in more productive crops which has been accelerated by ___________ of genes.

direct transfer

Plant transgenesis

direct transfer of genes to plants

Example of plant transgenesis

development of plant vaccines, plants that produce their own pesticides and are resistant to herbidices

What was the significant portion of several key crops in the US in 2018 that were transgenic?

94% of soybeans, 92% of corn, 90% of cotton

What percentage of GMOs are now present in conventional processed food in the U.S?

75-80%

Conventional “traditional methods” involve:

-selective breeding and hybridization

Cloning (PTC) “modern methods” include:

-protoplast fusion w/ plant tissue culture (PTC)

-leaf fragment technique and Ti plasmid w/ PTC

-gene guns w/ PTC

-chloroplast engineering w/ PTC

-antisense technology

How do “traditional methods” work?

sexual cross between two lines and repeated backcrossing between hybrid offspring and parent (can take years)

Polyploid plants (multiple chromosome)

-increases desirable traits, especially size

-using colchicine followed by hybridization, whole chromosomes can be transferred rather than single genes; can result in larger fruits and vegetables

Cloning “modern methods”

growing plants from a single cell w/PTC

What is the fusion of two protoplast cells from different species?

protoplast fusion

what is a protoplast?

a plant cell whose cell wall has been dissolved by the enzyme cellulase

What does polyethylene glycol create in the fusion of the two protoplast cells?

a cell that can grow into a hybrid plant

PTC Step 1 involves:

cytokinin and auxin present in equal levels = undifferentiated callus

PTC Step 2 involves:

more cytokinin and less auxin = growth of shoot buds

PTC Step 3 involves:

less cytokinin and more auxin = root formation

Leaf fragment technique & Ti Plasmid w/PTC

-small discs are cut from leaf

-cultured in a medium containing genetically modified (Rhizobium radiobacter) AKA: Agrobacterium tumefaciens

-transfected leaf discs are treated with plant hormones to first stimulate callus formation, then shoot and root development

Cultured in a medium containing genetically modified (Rhizobium radiobacter) AKA: Agrobacterium tumefaciens

-a soil bacterium that infects dicot plants ONLY

-R. radiobacter contains a plasmid, called Ti plasmid, that can be genetically modified

-T-DNA (T=transfer) from the Ti plasmid integrates with DNA of host cell

What are Gene Guns used for?

to blast tiny metal beads coated with DNA into an embryonic plant cell; animal cells too. (ex: tungsten, gold, titanium, or silver)

What are gene guns aimed at?

the nucleus or the chloroplast

What are marker genes used for?

to distinguish genetically transformed cells

-antibiotic resistance

What is the gene guns technique useful for?

useful in plants that are resistant to R. radiobacter Ex. Monocot plants

What does chloroplast engineering do?

-DNA in chloroplast can accept several new genes at once

-high percentage of genes will remain active

-chloroplast DNA is completely separate from genomic DNA

-no chance that transformed genes will be carried on wind to distant crops via pollen

Polycinstronic mRNA is present in:

prokaryotes

Polycinstronic mRNA contains:

codons for more than 1 gene (cistron)

Polycinstronic mRNA is transcribed from:

more than 1 protein

Polycinstronic mRNA has:

many initiation (start) and termination codons

Monocistronic mRNA is present in:

eukaryotes

Monocistronic mRNA contains:

codons of a single gene (cistron)