Bacteriology test 1

Prokaryotes vs Eukaryotes

Prokaryotes: single celled, no nucleus, simple chromosome structure, divides by binary fission, peptidoglycan cell wall, one type of ribosome

Eukaryotes: nucleus and membrane bound organelles, formed by bacteria and archaea relationship, less permeable cytoplasmic membrane with sterols, cell division my mitosis or meiosis, chitin or cellulose cell wall,

Bacteria vs archaea

Bacteria: larger domain, some photosynthetic, none form methane, medically important, membrane lipids- ester linked, flagella grow at tip, contain peptidoglycan

Archaea: smaller domain, not photosynthetic, none are known pathogens, occupy extreme habitats, methane formers, membrane lipids - ether linked, flagella assemble at base, contain pseudomurein,

Gram positive vs gram negative bacteria

Gram positive: thick peptidoglycan layer! have mesosome, surface proteins, stain purple for gram stain

Gram negative: thin peptidoglycan layer! Lipopolysaccharide layer with high amounts of endtoxins. pili, periplasmic space as well as cytoplasmic space, porin proteins as outer membrane, stain red or pink for gram stain.

Adhesion to host cells is from

pili, proteins, teichoic acid

immune recognition by host

all outer structures and secreted proteins create immune response from host

how do bacteria escape host cell recognition

antigenic variation, m proteins, capsule

how are bacteria adhesive?

pili, outer surface proteins

Controls for protein synthesis

• DNA

• transcription- mRNA synthesis

• protein synthesis

• inducers- IPTG, Arabinose

• Repressors

• Nutrients/ energy source

lac operon

Lactose metabolism. The genes in the operon allow bacteria to process lactose.

Cloning of foreign DNA for expression

several different types of plasmids, antibiotics, inducers, and bacterial strains

Plasmid features

1. Origin of replication

2. Promoters- inducible or constitutive

3. structural or fusion genes

4. ribosome binding sites

5. multiple cloning sites

6. transcriptional terminator

7. antibiotic resistance markers

How will you exploit a plasmid to modify what a bacterial cell does?

You can clone foreign DNA for expression! Have plasmid + “foreign DNA”. Knick plasmid and foreign DNA using restriction endonuclease. Add DNA to plasmid, seal with ligase to form hybrid plasmid which now modifies what the bacterial cell does.

Rolling circle replication

Replication initiator protein binds to origin of replication to nick the 5’ end so DNA polymerase III can bind to 3’ and replication can begin when helicase binds to prevent tangling of DNA. The original initiator protein will cut the strand once finished and ligase will fix the nick. Once strand is complete a loop is formed and RNA polymerase will bind and form complementary strand

Purification of recombinant protein

1. identify protein of interest

2. identify source of protein

3. identify gene that encodes protein

4. amplify gene

5. clone amplicon using PCR

6. transform bacteria to identify plasmids

7. reclone gene to expression plasmid

8. transform expression host

9. express recombinant protein

10. purify protein

11. use protein

Purposes to make recombinant protein

• to get information about protein

• difficult to isolate and purify protein in native organism

Bacterial population benefits from phages

• exposure drives population diversification

• phages can function as weapons against foreign bacterial populations

• facilitate genetic material transfer via horizontal gene transfer

• phages that are released by prophage induction interact with cells of metazoan host which can lead to expression changes

practical application using phages and process

Phages can be used to neutralize or block antibodies against a virus (example SARS- CoV-2)

• Infect mice with COVID 19, they produce antibodies

• take the b cells from infected mice and isolate the mRNA from the b cells then amplify by PCR.

• Clone PCR fragments into phage and then the phage infects bacteria to express heavy and light chains of cloned particles.

• Do multiple times until desired phage is acquired

• convert phage DNA to circular plasmid

• Insert plasmid into bacteria transform bacterial cells to have the Antibodies

 On your trip to Hawaii you collected some soil samples and plan to identify a new bacteria- how will you do it

• Collect soil

• Dilute soil

• lawned on plate

• isolate colonies

• lawn with target bacteria

• added isolated colonies to plate

• observe for zone of inhibitions

• if yes then microbe for soil sample produced antimicrobial against target bacteria

List specifically what recombinants proteins will you make example a specific cheese

• identify protein

• identify source

• identify gene encoding protein

• amplify gene

• clone amplification in PCR cloning vector (add distinguishing factor like antibiotic resistance)

• transform e. coli to make plasmids that hold the gene for specific cheese (in order to select e. coli containing plasmid add to plate that contains the antibiotic target)

• reclone gene of interest in expression plasmid

• transform expression host ( the bacteria that reads expression plasmid) to express recombinant protein ( the flavor of cheese)