Quiz #5: 11B, 12, 13, 14, and 15

Question 1

Lifecycle of helper T cell recognition

Answer 1

Th cell recognizes antigen bound to MHC class II of a B cell or macrophage

Th cell produces cytokines that direct further immune response

Question 2

What was KZH in the T cell experiment?

Answer 2

KZH was a T helper hybridoma, created by fusing normal helper T cells from mice immunized with antigen lysozyme + a T lymphoma cell with a reporter

Question 3

In T cell experiment, what reporter does KZH have and how does it work?

Answer 3

Gene lacZ (makes B-galactosidase) connected to IL-2 gene (makes cytokine IL-2) which is activated upon antigen binding. 
Cell is lysed to release B-gal using TritonX-100
B-gal now can now hydrolyze CPRG to cause color change

Question 4

In T cell experiment, what was the antigen and the antigen presenting cell?

Answer 4

Antigen is lysozyme

Antigen presenting was mouse B cell hybridoma LK35.2

Question 5

In T cell experiement, why did we culture the cells in 5% CO2

Answer 5

Because it established equilibrium with bicarbonate in growth medium, forming a buffer that maintains pH at about 7.4

Question 6

In T cell exp, what ingredients were in RPMI-1640?

Answer 6

L-glutamine, 
pyruvate, 
2-mercaptoethanol, 
HEPES, 
fetal bovine serum, 
antibiotics gentamicin & penicillin & streptomycin

Question 7

What temperature was T cell experiment plate incubated at day 2?

Answer 7

37ºC

Question 8

Narrow-spectrum antibiotics

Answer 8

effective only against a limited number of pathogens

Question 9

Broad-spectrum antibiotics

Answer 9

attack many different kinds of bacteria

Question 10

Cidal antibiotics

Answer 10

kill microorganisms

Question 11

Static antibiotics

Answer 11

Do not kill but inhibit growth

Question 12

Penicillin

Answer 12

First medically useful antibiotic

Produced by Penicillium notatum and Penicillium chrysogenum

Question 13

Streptomyces

Answer 13

Genus of Actinomycetes

Includes antibiotics: 
Streptomycin
Kanamycin
Vancomycin
Rifampin
Chloromycetin
Tetracycline

Question 14

What is an antibiotic that is now produced synthetically?

Answer 14

Chloromycetin is now chloramphenicol

Question 15

Applications of antibiotics

Answer 15

veterinary medicine, animal feed supplementation, plant disease control, food preservation

Question 16

Provide generic name, biological source, and MOA for beta-lactams

Answer 16

Penicillins and cephalosporins

Penicillium and cephalosporium

Inhibits synthesis of bacterial cell wall peptidoglycan

Question 17

Provide generic name, biological source, and MOA for macrolides

Answer 17

Erythromycin

Streptomyces erythreus

Inhibits rRNA associated with 50s LSU ribosome

Question 18

Provide generic name, biological source, and MOA for aminoglycosides

Answer 18

Streptomycin, neomycin, gentamicin

S. griseus, S. fradiae, Micromonospora purpurea

Inhibits 30s SSU ribosome

Question 19

Provide generic name, biological source, and MOA for tetracyclines

Answer 19

Tetracycline

Streptomyces aureofaciens

Inhibits binding of aminoacyl-tRNAs to ribosome

Question 20

Which antibiotic producing microbes did we use in 12A?

Answer 20

Penicillium notatum and Streptomyces griseus

Question 21

Which bacteria did we test in 12A and their reaction to each antibiotic?

Answer 21

E.coli: none P, some S
B.subtilis: full P, none S
S.aureus: full P, some S
Micrococcus luteus: full P, full S

Question 22

What temp were plates incubated at for 12A after days 1 and 2

Answer 22

Day 1- 30ºC

Day 2-37ºC

Question 23

What is the Kirby-Bauer method?

Answer 23

Inoculated Mueller-Hinton or TSA plates with antibiotic discs, measure inhibition zones

Question 24

Minimal inhibitory concentration (MIC)

Answer 24

The lowest concentration of antibiotic that will prevent growth, proportional to the diameter of the inhibition zone around the disc

Question 25

What method was used to standardize the disc diffusion bacterial inoculum turbidity and what value was used?

Answer 25

McFarland standards, 0.5 used

Question 26

What antibiotic discs were used in 12B?

Answer 26

Erythromycin (E15) Penicillin (P10) Tetracycline (TE30) Gentamicin (GM10)

Question 27

What bacteria were used in 12B?

Answer 27

E.coli and S.aureus at McFarland 0.5

Question 28

What stage of the bacterial growth curve is the bacteria most susceptible to antibiotics?

Answer 28

Log/exponential phase

Question 29

What are the intermediate values (in mm) of each antibiotic--- sensitive is anything more and resistant is anything less

Answer 29

E15: 14-17 P10: 12-21 (21-28 for staphylococci) T30: 15-18 G10: 13-14

Question 30

Horizontal gene transfer

Answer 30

Process where genes are transferred from one mature independent organism to another

Question 31

Conjugation

Answer 31

The process of genetic transfer that involves cell-to-cel contact between donor and recipient cells

Question 32

Transformation

Answer 32

free DNA is taken up by a cell and integrated into its genome

Question 33

Transduction

Answer 33

Transfer of genes between bacterial or archaeal cells by bacteriophages

Question 34

Competent cells

Answer 34

Able to take up free DNA

Question 35

What streptomycin resistant strain of bacteria was used in Ex. 13

Answer 35

Acinetobacter calcoaceticus

Question 36

Ex. 13 incubation temperature

Answer 36

30ºC

Question 37

Which of the 4 sectors on the plate should have no growth and why?

Answer 37

DNA only because there are no cells present

Question 38

What temperature was the DNA incubated at in Ex. 13 and why

Answer 38

60ºC to lyse cells and have purified DNA

Question 39

R factors

Answer 39

Conjugative plasmids the carry genes for resistance to antibiotics

Question 40

tra genes

Answer 40

Donor genes that encode proteins that help carry out conjugation

Question 41

F (sex) pilus

Answer 41

tra gene product that mediates contact between donor and recipient for conjugation

Question 42

In Ex. 14, what plasmid was being transferred? What are some key features of this plasmid?

Answer 42

``` pBBR1MCS-2: Kanamycin r gene lacZ gene mob multiple cloning site (MCS) ```

Question 43

Restriction endonuclease

Answer 43

enzymes that cut DNA into pieces at the restriction sites (in MCS region)

Question 44

What is the donor E.coli strain used in Ex. 14, what antibiotics is it sensitive/resistant to

Answer 44

Strain S17-1 containing tra genes for plasmid transfer. Sensitive to nalidixic acid, resistance to kanamycin

Question 45

What is the recipient E.coli strain used in Ex. 14, what antibiotics is it sensitive/resistant to

Answer 45

Strain DH5å Sensitive to kanamycin, resistant to nalidixic acid

Question 46

How did the three plates grow in exercise 14?

Answer 46

Nalidixic acid: recipient only, white Kanamycin: donor only, blue Nal+Kana: mate growth, blue

Question 47

Bacteriophages or phages

Answer 47

viruses that infect bacteria, basically a fragment of genetic material with protein coating

Question 48

Bacteriophage infection cycle

Answer 48

Phage binds via tail fibers to a cell receptor Phage genetic material, stored in capsid, is injected into host In host, gentic material either is used to make more phages and lyse cell (lytic) or forms prophage in genome and becomes latent (lysogenic)

Question 49

Phage titer

Answer 49

The concentration of infectious phage particles per mL of growth medium

Question 50

Plaques

Answer 50

clear zones in a lawn of bacteria where phages killed cells

Question 51

What broth were E.coli in in Ex 15?

Answer 51

Luria-Bertani (LB) broth

Question 52

What facilitates phage attachment to bacterial receptors?

Answer 52

magnesium ions

Question 53

Temperate bacteriophages and example

Answer 53

carried by host cells for generations-- lysogenic Lambda temperate bacteriophage infects E. coli, can enter lytic or lysogenic cycle tho

Question 54

Repressor protein cI

Answer 54

product of phage gene that regulates the lysogenic cycle of lambda phage, binds to operator region

Question 55

Regulatory protein Cro

Answer 55

promotes lytic cycle in lambda phage, binds to same operator as cI but opposite effects