Biotech Exam 3 BSC4422 FIU

*Microbial Biotechnology

Define Microbes & Name 4 Types?

Microorganisms are tiny organisms that are too small to see be seen individually.

Bacteria, fungi, algae, & protozoa

Less than __#__% of all bacterial species have been identified, cultured, and studied.

Bacterial species are estimated to comprise of over __#__% of Earth's biomass.

1%>

50%<

Define Bioprospecting & 2 Marine Examples?

the discovery & development of new products from biological resources.

Marine plankton (Plankton) & snails (Gary) = rich sources of antitumor & anticancer molecules.

*Microorganisms as Tools

Microbial Enzymes are used in applications from ______ production

to ____________ biology research.

food

molecular

3 Examples of microorganisms as tools?

1. Taq DNA Polymerase

-heat stabile, isolated from a thermophile

2. Cellulase

-makes animal food more digestible

-stone washed jeans

3. Subtilisin

-laundry detergents

Define Yeast Two-Hybrid System

technique to detect protein-protein interactions

Yeast Two-Hybrid System determines?

if protein A and protein B interact with each other

Yeast Two-Hybrid System

Protein of interest is labeled as ______ and is fused to a DNA binding domain protein (DBD) and binds to Promoter.

Proteins that bind to the bait (second protein of interest) are called ________ and are fused to an activation domain protein (AD) and binds to Enhancer.

Any protein that binds to the Bait will activate the transcription of a _______ reporter gene.

Bait

Fish

HIS

When protein A and B interact, the DBD and AD will be?

brough together

What is the result of an active transcription initiation complex that is made from the Yeast Two-Hybrid System?

expression of a specific reporter gene (ex: lacZ)

HIS gene allows for synthesis of _____

amino acid histamine

What is UAS?

Enhancer

Bait plasmid has gene that allows for the synthesis of ___________

Fish plasmid has a gene that allows for the synthesis of ________

Tryptophan

Leucine

*Video Yeast Two-Hybrid System

Steps -1-3:

1. Construct a bait plasmid & a library of DNA in the FISH plasmid.

*Each type of plasmid contains a selectable marker (i.e. essential amino acid)

2. Both types of plasmids are transformed into yeast cells

3. Cells are placed in an amino acid deficient medium

Why are the cells placed in an amino acid deficient medium?

so ONLY the ones that contain both plasmids will grow.

*Using Microbes for everyday applications

CRISPR stands for

Clustered Regularly Interspaced Short Palindromic Repeats

Define palindromic

a word that spells the same thing backward and forward.

Using Microbes for everyday applications:

CRISPR

1. ___________ immune system that provides acquired immunity

2. found in approximately ~______% of sequenced bacterial genomes

3. segments of DNA containing short, repetitive base sequences

4. repeats are palindromic

5. each repetition is followed by a short segment of spacer ______

6. spacer DNA is from previous exposures to foreign DNA

(example: virus or __________)

7. ____ gene are located next to CRISPR sequences

8. cas9 must be complexed with a _______ & a separate trans-activating crRNA (tracrRNA or trRNA) to cleave target DNA

1. Prokaryotic

2. 50

5. DNA

6. plasmid

7. cas

8. crRNA

CRISPR: Prokaryotic cell

(Powerpoint Slide 292)

cas12 gives _______ ends

CRISPR locus transcription results in pre-crRNA which is a _______________ type of structure

sticky

hair-loop pin

Editing a gene using CRISPR/CAS9 technique

Steps 1-4

1. Scientist create a genetic sequence called "guide RNA" that matches the piece of DNA they want to modify.

The guide sequence corresponds to ________

1. Spacers

Editing a gene using CRISPR/CAS9 technique

Steps 1-4

2. This sequence is added to a cell along with a protein called Cas9, which acts like a pair of __________ that cut DNA

2. scissors

Editing a gene using CRISPR/CAS9 technique

Steps 1-4

3. The guide RNA homes in on the target DNA sequence, and Cas9 ______ it out.

Once their job is complete, the guide RNA and Cas9 ________ the scene.

3. cuts /

leaves

Editing a gene using CRISPR/CAS9 technique

Steps 1-4

4. Now, another piece of DNA is swapped into the place of the old DNA and enzymes __________ the cuts.

4. repair

CRISPR cas: ____________________

Cas9: DOES the actual cutting

-WHERE we have to cut

*Microbes for Food Applications

Food Products

_________ are used with traditional & modern biotechnology to make many foods

Microbes

4 Examples of Food products produced:

1. bread

2. yoghurt

3. cheese

4. alcohol

What was the first recombinant DNA food ingredient approved by the FDA in 1999?

a recombinant form of an enzyme used to make cheese

Traditionally:

Curds to make cheeses are made from _________,

an enzyme which is extracted from the stomach of calves.

rennin

What was the name of a rennin that was cloned and expressed in bacteria and is LESS expensive and easier to produce?

Chymosin

Fermentation ≠ ____________

a. energy from sugars

b. bioreactors

b. Bioreactors

Fermentation is the process of deriving energy from sugars in the absence of _________.

oxygen

Lactic acid fermentation used to make? Name 6 Examples:

Alcohol fermentation used to make? Name 3 Examples:

1. yoghurt

2. sour cream

3. cheese

4. bread

5. sauerkraut

6. vinegar

1. beer

2. wine

3. champagne

*Therapeutic Proteins

________ are used to produce medically important proteins.

Bacteria

Genetically engineered in 1978, What was the first recombinant molecule expressed in bacteria for use in humans?

Insulin

What year was Humulin commercially available by Genentech?

1982

*Vaccines

First vaccine developed in 1796 by _____.

Edward Jenner

Dr. Jenner used LIVE cowpox virus to vaccinate James Phipps (farmer's son) against ___________

SMALLPOX

Dr. Jenner was an English Physician & ________.

He was known as the Father of __________.

Scientist

Immunology

*Immune System & Antibodies

Define Antigen? 6 Examples?

foreign substance that stimulates an immune response

Examples:

1. whole bacteria

2. fungi

3. viruses

4. proteins

5. lipids

6. carbohydrates

Immune system responds to antigens by producing ________.

This is called __________-_________ immunity.

antibodies

antibody-mediated

______ cells recognize and bind to the foreign antigen.

^ These cells, with the help of T helper cells, undergo activation to form ______ cells that produce antibodies.

B

plasma

Antibodies are:

a. Specific

b. Non-specific

Macrophage can then recognize the antigens coated with _______ and phagocytize them.

a. Specific

antibodies

True or False

Sometimes our natural production of antibodies is not enough to protect us from pathogens

TRUE

What has parts of a pathogen or whole organism that can be given to humans or animals by mouth or injection to stimulate the immune system against infection by those pathogens?

Vaccines

4 Types of Vaccines:

1. What is a subunit vaccine made from?

2. What vaccine uses LIVE viruses or bacteria that have been weakened through aging or altering growth conditions to prevent replication?

3. Inactivated (killed) vaccines are made by?

4. mRNA vaccines use non-replicating mRNA encased in a lipid nanoparticle coat so that it can _____ the cells. (ex: Moderna, Pfizer, BioNtech)

1. injecting portions of viral or bacterial structures (i.e. purified proteins)

2. Attenuated

3. killing the pathogen and using the dead microorganism for the vaccine

4. enter

Currently, a majority of _____vaccines are made using ____ approaches in which the vaccine is produced in _____.

subunit; recombinant DNA approaches; microbes

2 Main subunit vaccines are:

1. _____

2. _____

1. Uses genes for proteins on the outer surface of virus are cloned into plasmids and transformed into yeast. Fusion proteins are produced by the yeast that are purified.

2. Protects against 9 strains of HPV

1. Hepatitis B

2. Gardasil

Biotechnology companies are working on over 50 targets for vaccine development including?

Influenza, Tuberculosis, Malaria, HIV, and Covid-19

What is the importance of sequencing microbial genomes?

1. Find new strains

-bioremediation

-identify disease causing organisms

2. To identify genes involved in

-bacterial metabolism

-cell division

-genes that cause human and animal illness.

What causes ear and lung infections that kills 3 million children worldwide each year?

Originally had low efficacy on children.

Streptococcus pneumoniae

What year was the S. pneumoniae genome sequenced and many genes encoding proteins on the surface of the bacteria were discovered?

2001

What is metagenomics?

study of genetic material recovered directly from environmental samples

What is a microbiome?

collection of genome of microorganisms in a particular environment on or in the human body

What year did the NIH announce the Human Microbiome Project?

2008

Human Microbiome Project was a ____ year project used to sequence over ____ genomes of microorganisms.

Over ______ genomes have been catalogued as of 2019.

5

600

2200

Human Microbiome Project goals:

A) determine if individuals share a core human microbiomeB) understand how we acquire & maintain microbial communitiesC) understand how changes in the microbiome can be correlated with changes in health & conditions that affect the microbiomeD) develop new methods for analysis of the microbiomeE) address ethical, legal, and social implications raised by human microbiome research

Microbial Diagnostic is a technique to?

detect and track microbes

What are the 3 covered bacterial detection strategies? (ex: used to detect and track bacterial contamination of food)

1. DNA sequencing

2. PCR

3. RFLP

(restriction fragment length polymorphism)

Microarrays has created new approaches for detecting and identifying pathogens and for examining host responses to infectious diseases, where _______ species tested in parallel.

50,000

Microarray is used to find "____" changes in gene expression for a particular pathogen

signature

The world population has nearly ____ in the past _____ years while arable land has only increased by ____ %.

doubled

40

10%

What has enabled plant geneticist to feed larger numbers of people?

a. Improved crop breeding

b. Direct transfer of genes

What has accelerated development of new, more productive crops?

a. Improved crop breeding

b. Direct transfer of genes

a. Improved crop breeding

b. Direct transfer of genes

What is the direct transfer of genes to plants?

(ex: development of plant vaccines, plants that produce their own pesticides and are resistant to herbicides)

Plant transgenesis

In ______, 13.3 million farmers in ____ countries planted transgenic crops. (90% in developing countries)

2008; 25

In 2018 there a significant portion of several key crops in US that were transgenic?

____% of soybeans

____% of corn

____% of cotton

94

92

90

What category do these 5 fall under?

A) Protoplast fusion w/ plant tissue culture (PTC)

B) Leaf fragment technique & Ti Plasmid w/ PTC

C) Gene guns w/ PTC

D) Chloroplast engineering w/ PTC

E) Antisense technology

cloning

What is between 2 lines and repeated backcrossing between hybrid offspring and parent that can take years?

Sexual cross

What increases desirable traits (size) and uses colchicine followed by hybridization, whole chromosomes can be transferred rather than single genes and can result in larger fruits and veggies?

Polyploid plants (multiple chromosomes)

Cloning is the process of growing plants from __#__ cell(s)?

a. 1

b. 2

c. 10+

a. 1

A) Protoplast fusion w/ plant tissue culture (PTC)

is the fusion of two protoplast cells from different _____?

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

species

Protoplast

What creates a cell that can grow into a hybrid plant (ex: broccoflower)

Fusion of two protoplast cells using polyethylene glycol

If cytokinin & auxin present in equal levels=

high cytokinin & low auxin causes growth of ________

low cytokinin & high auxin causes ______

undifferentiated calluses

shoot buds

root formation

B) Leaf fragment technique & Ti Plasmid w/ PTC

method used in plant transgenesis uses small discs cut from a leaf that are cultured in a medium containing genetically modified Rhizobium radiobacter.

R. radiobacter is a soil bacterium that infects _______ plants ONLY, contains a plasmid called Ti plasmid that can be genetically modified, TDNA from the plasmid integrates with DNA of host cells.

dicot

Transfected leaf discs are treated with plant hormones to first stimulate _____ then _______.

callus formation;

root & shoot development

Gene guns are used to blast tiny metal beads coated with DNA into an embryonic plant (aimed at the ______ or chloroplast) and animal cells. (hint: this technique is useful in plants that are resistant to R. radiobacter like ________ plants)

nucleus

Monocot

What is used to distinguish genetically transformed cells for antibiotic resistance?

Marker Genes

Transgenic Plants

Cloning Type #3

Chloroplast engineering

True or False

Chloroplast DNA is completely separate from genomic DNA

TRUE

DNA in chloroplast can accept ______ new genes at once, where a high percentage of genes will remain active.

a. only 1

b. several

several

Is it possible for transformed genes to be carried on wind to distant crops via pollen?

Not possible

What type of mRNA is present in prokaryotes and contains codons for more than 1 gene (cistron), transcribed from more than 1 gene, and codes for more than 1 protein?

a. Polycistronic mRNA

b. Monocistronic mRNA

a. Polycistronic mRNA

Polycistronic mRNA has ____ initiation & termination codons.

a. none

b. 1

c. many

c. many

What type of mRNA is present in eukaryotes, contains codons of a single gene (cistron), transcribed from a single gene, and codes only for a single protein?

Monocistronic mRNA

Monocistronic mRNA has ____ initiation & termination codon.

a. none

b. 1

c. many

b. 1

Which mRNA targets Streptococcus mutans?

a. Monocistronic mRNA: Guy's 13 monoclonal antibody

b. Polycistronic mRNA: Bt Cry Operon

a. Monocistronic mRNA: Guy's 13 monoclonal antibody

Transgenic Plants

Cloning Type #4

Antisense Technology

What does Antisense technology insert into a cell?

a complementary copy of a gene

In antisense technology, genes encode an mRNA molecule called _____ molecule, that bind to normal mRNA (sense molecules) and inactivate it. (ex: Flavr Savr tomato)

antisense

What are the practical applications of Antisense technology?

a. vaccines for plants & herbicide resistance

b. genetic pesticides & enhanced nutrition

c. increase growth development & immunity

d. pharmaceuticals to fuel

e. A, B, & D

f. A & B

g. All of the above

e. A, B, & D are correct

Vaccines for Plants

Where is a vaccine encoded in a plant?

in it's DNA

What does TMV stand for? (hint: a gene from this virus is inserted into tobacco plants, producing protein that stimulate the plants immune system & makes it resistant to virus)

Tobacco Mosaic Virus

Genetic Pesticides

What is the name of the bacterium that is a natural pesticide (used for over 100 years), that produces a protein that kills harmful insects and their larvae? (hint: Also known as Bt.)

Bacillus thuringiensis (Bt)

Genetic Pesticides

What is the name of the genes that can be inserted into a plant's DNA, which crystallized proteins fuses to gut lining of insects and creates a built in defense against certain insects? (hint: Controversy around Monarch butterflies)

Bt. Cry

(Bacillus thuringiensis)

Herbicide Resistance

What is the problem with using traditional week killers?

They eliminate desirable plants along with the weeds

Herbicide Resistance

What are the pros of herbicide resistance?

1. can genetically engineer _____ to be resistant to common herbicides

2. allows farmers to control ______ with chemicals that are milder and more environmentally friendly than typical herbicides

1. crops

2. weeds

Herbicide Resistance

When there is a resistance to glyphosate, the enzyme EPSPS is blocked and causes the plant to die because?

this is a key biochemical pathway in plants.

True or False

Crops that transgenically produce an alternative enzyme are not affected by glyphosate. (hint: resistance to glyphosate blocks enzyme EPSPS)

TRUE

1. What plant commonly contains herbicide resistance genes?

2. And have weeds evolved to be glyphosate resistant?

1. Soybeans

2. Yes

Enhanced Nutrition

What has been engineered to contain large amounts of beta carotene and what does the body convert this to?

Golden Rice

Vitamin A

What year & Why did farmers stop planting golden rice?

2011; concerns from environmental orgz.

What can be the ideal protein factory and is used to grow medicine?

Plants

Why are plants the ideal protein factory? (hint: we use them to grow medicine)

Inexpensive, edible vaccines, do not need refrigeration