Lock In Micro 3

Immunity

Phage enters and having a defense mechanism to fight off infection

Resistance

Phage cannot enter so no infection

What structural difference is there between immunity and resistance?

Immunity has phage receptor, resistance has none

Lysogen immunity

Phage integrates its genome into bacteria, which contain repressor proteins that shut off the lytic function to keep the phage in. This prevents infection of the same genome

How do bacteria block phage from binding?

Use of masking phage receptors (glycosylation, proteins, modification, omv)

What is OMV (outer membrane vesicle)?

Defense mechanism of bacteria where it sacrifices a piece of its membrane as decoys for phage to bind to

What do bacteria target when phage injects genome? (3)

degradation of the phage genome

abortive infection

inhibition of replication

Restriction Modification Systems (RM)

Higher plaquing on field means higher infection and less cleaving by endonuclease

How do bacteria degrade phage DNA?

Endonuclease

How does bacteria distinguish between self and nonself?

Bacteria DNA are methylated, nonself is nonmethylated

Where do bacteria cut DNA?

Specific palindromic recognition sequences

What are the two ways endonuclease cuts, and which one is easier to bind back?

Blunt cuts - not easy to recombine because no single strand guide

Staggered cuts - easy to recombine because has single strand guide

What type of system is RM?

Type II

In CRISPR how does bacteria recognize phage?

Phage contains PAM which tags the phage as nonself to be recognized by Cas nuclease

Where do Cas proteins bind on phage genome?

Gene adjacent to PAM site

Where are Cas proteins made?

By host in Cas operon of its gene

Where do Cas integrate phage DNA?

Between palindromic repeats in chronological order

What is included in the spacer?

Only gene adjacent to PAM site

CRISPR: After integration of phage gene, what happens after transcribing the gene?

Formation of crRNA that has repeats as the stem and loop

CRISPR: what binds to Cas nuclease and what happens?

crRNA binds and help guide to complimentary DNA by PAM site of phage to cut both strands

Which domain of life has CRISPR?

Most Bacteria and some archaea

Abortive infection system (eukaryotes)

interferon-mediated and if infected induces apoptosis to be eaten by leukocyte

Abortive infection system (prokaryotes)

Prevents replication of phage by translating PICI gene which modifies the capsid head to be smaller, making the phage gene unable to enter but rather the PICI DNA is in the capsid

Spreads defense to nearby bacteria

PCR equation

Different DNA restriction fragments cut with the same endonuclease can … in random order, regardless of their original source

anneal

How to seal up ends of DNA?

How to clone DNA without PCR?

Use plasmid cloning vector and foreign DNA that is partially chopped by endonuclease and mix it before annealing back together with ligase

What is the con of using plasmid cloning vectors ?

How to prevent annealing in DNA?

Removal of phosphate group using phosphatase

What happens if you mix digested and phosphatase-treated vector DNA with inserted DNA digested with the same nuclease?

DNA is partially ligated with 2 breaks because each strand is missing a phosphate group to bind with OH

OH + P

Seal

Only OH

No seal

Ideal cloning vector has… (3)

Selectable marker

Many cloning sites within a second marker

high copy number

Second marker

Used as ID for plasmids that contain many cloning sites

Cloning vector EX: blue vs white colonies

Blue: cleavage of xgal by lacZ protein, meaning that the protein is functional

White: no cleavage of xgal by lacZ protein, meaning that the protein is nonfunctional

Reporter genes are created by…

transposons

Reporter genes

Used to know and manipulate how a gene is regulated, where we know the product of

Where/How are transposons integrated on a gene?

Inverted repeats that set the ends of transposons, transposase recognition site

Are transposons LOF or GOF

LOF

Reporter gene in the middle of lacZ: If there are 2 terminators and 2 ribosomal binding site on a gene, how many RNA/Proteins are made?

2 (1 functional = lacZ, 1 nonfunctional)

Normally, reporter gene are under control of … promotor

Another gene’s

Transcriptional reporters are created by…

deletion of the terminator and promoter upstream of reporter gene

Reporter genes in middle of lacZ: If there are 1 terminator and 2 ribosomal binding sites, how many RNA and proteins are made?

1 RNA, 2 proteins (1 non functional, 1 functional = lacZ)

Reporter gene in middle of lacZ: what do we measure to tell us the activity of the promoter of the target gene (lacZ)?

reporter gene protein

Reporter gene in middle of lacZ: If there is 1 RBS and 1 terminator on the gene how many RNA/proteins are made?

1 RNA, 1 protein (functional reporter fusion!)

What can translation fusions tell us?

translational regulation and localization of a gene

EX of localization from reporter genes

Formation of green fluorescent protein translational fusion with FtsZ in E Coli

tracrRNA

accessory RNA that binds to the repeat portion of CRISPR gene, handle for Cas9 protein to recognise

How did Charpentier and Doudna change tracrRNA?

Rather than needing 2 units for crRNA, formation of single guide RNA (sgRNA) only had 1 unit of crRNA (optimized)

How many nuclease motifs does Cas9 have and what does it do?

2 to cleave specific strands of DNA

CRISPR NHEJ (Non-Homologous End Joining)

After cleavage with Cas9, DNA tries to repair itself, filling in gaps with insertions or deletions

Causes LOF

CRISPR Allelic replacement

After cleavage with Cas9, an alternative allele nearby will be repaired and replace the gaps

Causes homologous recombination via with wild type allele

CRISPR Cas9 can repress… by

Transcription by using dCas9 and sgRNA to bind to complementary (dead and cannot cut)

CRISPR Cas9 can activate… by

Transcription by having an activator protein on Cas9

CRISPR Deamination of A turns it to… that bind with

Inosine that binds with Cytosine

CRISPR Deamination of C turns it to… that bind with

Uracil that binds with Adenine

How is base editing achieved in Cas9?

Fusing A or C deaminase (protein) to a modified Cas9, which changes the template nucleotide before cutting and repairing

Innate immunity (4)

Present at birth

Barriers to infection

Specialized cellular defenders

Nonspecific responses to target all microbes

What is the oldest immune defense? Innate or Adapted?

Innate

Innate is found in all…

Eukaryotes

What are considered first line defense in innate?

Physical and chemical defense that prevent entry to sterile tissue

What is considered second line defense in innate?

Chemical and leukocytes that detect presence of microbes in sterile tissue

What are considered physical barriers? (2)

Skin, mucous membrane

Why is skin a good physical barrier? (3)

Tight connective tissue that prevents microbial entry

Regeneration of epithelial cells that push out microbes

Dead cells fall off that remove bacteria on it

Why is skin a good chemical barrier?

Acidic, dry, and salty disrupts bacteria membrane

Defensins

Antimicrobial peptides that is (+) charged which disrupts the (-) interactions of bacterial membranes

What are the 2 classes of vertebrate defensins?

Alpha and beta defensins

Alpha defensin

Stored in cytoplasmic granules of neutrophils

Beta defensin

Found in secretions at mucous membranes

Why is the mucous membrane good physical barrier?

Tight junctions that prevent entry

Bathed in secretions that move bacteria away from surface

Why is the mucous membrane good chemical barrier?

Acidic and deprives nutrients (Fe) from bacteria

What happens when bacteria enter the skin via cut?

Cells sense microbes and alert via releasing cytokines that recruit phagocytes to the site to eat up microbes

How do our white blood cells sense microbes?

Use of Pattern Recognition Receptors and Cytokines

MAMPs (Microbe-associated molecular patterns)

Microbe structures have tags on bacterial structures as foreign

What are examples of MAMPs

Peptidoglycan, LPS, Teichoic acid, Flagellin, f-Met peptides

What recognizes MAMPs? (2)

Toll-like or NOD-like receptors on host cell

Pattern recognition receptors (PRRs)

Alarm systems that activate when there is an intruder

Location of NOD-like

Sense microbes within the cell

Location of toll-like

Outside the plasma membrane

What happens when MAMPs are detected or bound by PRRs in or out the cell?

Signaling cascades in the cell that activate proteins (cytokines) that act as an alarm to neighboring cell

What are the eyes and ears of the immune system?

PRRs

What are the alarms of the immune system?

Cytokines

What are the two main phagocytes that clear infections?

Macrophage and Neutrophils

Macrophages (3)

In tissues all over the body

Monocytes in blood differentiates when enters to infection site

Long term

Neutrophils (3)

Found in blood and sites of infection

Short term

Contain granules

Is in a Phagolysosome

Contains reactive oxygen species that kill the bacteria, and digestive enzymes (proteases, lysosomes, defensins) that degrade it

What is in the reactive oxygen species (3)

Superoxide, peroxide, hypochlorite

What two fuses to form a phagolysosome?

Phagosome and lysosome?

Neutrophils engulf microbes by…

Phagocytosis

What can neutrophils throw?

Neutrophil extracellular traps around nearby pathogens

What happens when neutrophils eat bacteria?

Undergo NETosis which spews chromatin that contains antimicrobial (+) compounds towards bacteria

What are the 2 specialized immune cells?

Macrophages and neutrophils

Immune system

Systems of organs, tissues, cells, and cell product

Adaptive immunity

Adapts throughout the organisms life

Specialized cellular defenders

Reactions to antigens

Has memory

What is an antigen

Chemical or protein that is foreign to the body that causes an immune response

What is the memory in immune response?

B and T cells

Where can you find B and T cells?

Through out the body in blood

Where can you find macrophages and dendritic cell?

Peripheral tissues

Dendritic cell

Digest bacteria and presents components on the surface of cells and loaded in MHC