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MBB265 > Semester 1 > Flashcards

Semester 1 Flashcards

1
Q

Typically how long are covalent bonds in proteins, and what makes these distances shorter?

A
  • 1.2-2.1Å
  • C-C ≈ 1.5Å
  • shorter if double/triple bond character involved
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2
Q

Typically how long are hydrogen bonds in biological systems?

A
  • 2.6-3.2Å
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3
Q

Where in protein can hydrogen bonds be formed?

A
  • between main chain atoms
  • between main and side chain atoms
  • between main/side chain atoms and surrounding molecules in solvent, eg. water or substrates/products
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4
Q

Hos is it poss for diff protein chains to adopt distinct conformations and shapes?

A
  • polypeptide backbone possesses inherent flexibility, as rotations poss about certain bonds in main chain
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5
Q

What 2 angles in the main chain can vary?

A
  • phi = rotation around bond joining α C to peptide N

- psi = rotation around bond joining α C to carbonyl C

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6
Q

What 3 groups can conformation of polypeptide at each residue be classified into?

A
  • allowed = all non-bonded interactions favourable
  • generously allowed = few poor steric interactions, so observed in real structures
  • disallowed = adverse steric interactions are such that these conformations are rarely, if ever, observed
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7
Q

When might ‘disallowed’ conformations be allowed?

A
  • some exceptions where other favourable interactions offset energy penalty paid for being in disallowed region, eg. specific functional role
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8
Q

What is the polarity of the polypeptide chain?

A
  • N –> C ter
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9
Q

Why are 3^10 helices less energetically stable as α-helices?

A

tighter helix so…

  • non ideal H-bonds
  • pot sidechain clashes
  • loss of internal packing
  • less favourable phi and psi angles
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10
Q

Is Serratia gram +ve or -ve?

A
  • -ve
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11
Q

Why is Serratia a good model for opportunistic pathogens?

A
  • (mostly) harmless so safe in lab
  • visible phenotype (red pigment), which is quite rare for bacteria
  • performs many of same functions as other opportunistic pathogens, eg. S. aureus
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12
Q

What are pathogenic Serratia species almost always assoc w/?

A
  • nosocomial (hospital acquired) infections

- or intravenous drug users

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13
Q

What is the key feature which makes Serratia adaptable as an opportunistic pathogen?

A
  • ability to reg gene expression in response to changes in env factors
  • once encounters host, changes in temp and nutrient availability (eg. iron) sensed by cells (Quorum sensing) and virulence factors can be prod in response
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14
Q

What is the role of prodigiosin (Pig) in Serratia?

A
  • antibiotic w/ antibacterial, antifungal and antiprotozoal activities
  • 2° metabolite –> no function in cell growth
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15
Q

Where is Pig found in Serratia, and why?

A
  • hydrophobic, so tends to remain assoc w/ cell membrane
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16
Q

Apart from Pig, what other 2° metabolite does Serratia prod, and how does it differ?

A
  • carbapenem (Car)
  • β-lactam antibiotic
  • more hydrophilic than Pig
  • secreted from cells into env and is therefore diffusible
  • made by another set of enzymes in complex biosynthetic pathway
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17
Q

How may Pig and Car allow Serratia to compete better?

A
  • killing off other non-Serratia bacteria in env
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18
Q

What are the most important virulence factors made by Serratia?

A
  • damaging secreted enzymes, inc proteases and DNAses, which degrade host tissue macromolecules, releasing nutrients to allow growth
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19
Q

How can some species of Serratia also be pathogenic to plants?

A
  • prod pectinases and cellulases that break down plant cell wall
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20
Q

When are all antibiotics and extracellular enzymes of Serratia made in large quantities?

A
  • stationary phase, when cell density reached high levels
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21
Q

What is Quorum sensing, and how does it work?

A
  • special control system
  • switches on genes for antibiotic and virulence factor prod in response to chemical signal or autoinducer (AHL) prod by individual cells
  • AHL only reaches threshold conc at high cell densities in a closed culture, as every cell contributes small amount of signal
  • threshold activates receptor DNA BP, that can bind target gene promoter and activate transcrip of genes
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22
Q

Why does Serratia delay the synthesis of virulence factors until high no. of cells?

A
  • activation at low pop density would not provide high enough autoinducer conc to overpower host defense mechanism
  • so would stimulate host defence response, giving host an adv over bacteria
  • so delay guarantees survival
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23
Q

What are the 2 key components of quorum sensing in Serratia?

A
  • enzyme that makes AHL

- regulatory binding protein, to enhance transcrip of target genes when AHL bound

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24
Q

What are 2 examples of systems under the control of quorum sensing in Serratia?

A
  • pigment prod

- swarming behaviour

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25
Q

How can you demonstrate quorum sensing in Serratia on a streak plate?

A
  • streak WT Serratia next to indicator strain
  • indicator strain is mutated version of C. violaceum (colourless)
  • watch bacteria change from white to purple when WT growing nearby
  • inc LIS mutant as eg. of Serratia mutant defective in quorum sensing
  • LIS mutant did not cause indicator strain to turn purple
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26
Q

What is a pleiotropic mutation, and what can they indicate?

A
  • mutations affecting 2 or more phenotypes

- indicate regulatory connections

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27
Q

How was Serratia mutagenised for our mutant screens?

A
  • transposon mutagenesis
28
Q

How could you design an indicator plate for detection of a new phenotype in Serratia?

A
  • need simple assay you can repeat for 1000s colonies, eg. cellulose
  • cellulose insoluble so appears cloudy
  • sugars soluble so appear clear
  • stab colonies onto plate containing cellulose
  • colonies secreting cellulose will have clear halo (of sugar) around them
  • cellulose-deficient mutants lack halo and are selected
29
Q

What can a cross feeding experiment tell you?

A
  • order of action of genes in a pathway
30
Q

What is cross feeding?

A
  • if 2 mutants w/ diff blocks in pathway are placed close together on an agar plate
  • diffusion of biosynthetic intermediates accum in these mutants may be able to restore, eg. red pigment, prod in one or both mutants
  • ie. use external supply excreted into medium instead of normal conversion pathway
31
Q

How does cross feeding show the order of genes?

A
  • identify feeder (colourless)
  • identify being fed (red)
  • feeder must act later in pathway than being fed
32
Q

In a cross feeding experiment what acts as the control?

A
  • middle part of streak not near another mutant
33
Q

What would a pleiotropic mutant look like if defective for pigment, carbapenem and AHL production in Serratia?

A
  • white (lacks pigment)
  • lack purple halo on AHL plate
  • lacks halo of ESS (carbapenem) plate
34
Q

When might cross feeding not work?

A
  • if accum metabolic intermediates were toxic to cell
  • if intermediates could not diffuse through media or travel across cell membrane
  • if metabolic pathway not linear (ie. branched), would be imposs to determine order of action of genes
35
Q

What was the nature of the Serratia LIS mutant, and how do we know this?

A
  • prevents prod, but not detection, of AHL
  • can detect it as cross feeding from WT to LIS
  • but cannot prod it itself
36
Q

If LIS mutant is colourless, what does this suggest about control of Pig synthesis in Serratia?

A
  • LIS can’t prod AHL, but when externally supplied colony turns red
  • suggests AHL req for upreg of pigment prod
37
Q

What is a poss mol basis for the pleiotropic Serratia mutants?

A
  • many systems inder quorum sensing control
  • mutant defective in AHL prod, detection or signalling may have multiple phenotypes (eg. Pig and swarming)
  • mutation could be in any of quorum sensing genes
  • upstream regulator of both quorum sensing and pig productions
  • mutations at this locus would prod pleiotropic mutants
38
Q

How can swarming behaviour of Serratia be demonstrated?

A
  • place on low % agar and observe colony behaviour
39
Q

What is swarming?

A
  • flagella driven coord movement of pop of bacteria across solid surface
40
Q

Why is it advantageous for opportunistic pathogens to swarm?

A
  • for tissue invasion
  • overcoming host defences
  • secretion of higher levels of extracellular enzymes and toxins
41
Q

How do hard and soft-agar swarmers differ?

A
  • hard-agar swarmers differentiate into specialised swarm cells that are elongated and have increased no.s flagella
  • soft-agar swarmers (eg. Serratia) generally don’t have differentiated morphology, movement often enabled by secretion of powerful extracellular biosurfactants, whose synthesis is under quorum sensing control
42
Q

Why is transposon mutagenesis unsuitable for isolation of mutants in essential genes, and what could be used instead?

A
  • KO function of gene
  • kills mutant so imposs to isolate/maintain/study
  • use conditional mutants, eg. Ts mutants, often due to point mutations prod by chemical mutagens like EMS
43
Q

What is an expression cassette?

A
  • part of vector DNA used for cloning and transformation
44
Q

What is the overall procedure we used for transformation experiment?

A
  • digest expression cassette in plasmid to isolate cassette
  • digest yeast LEU2 vector w/ same RE and treat w/ alkaline phosphatase (to avoid self ligation)
  • ligate and transform E. coli
  • identify and purify correct plasmid
  • digest w/ RE to linearise plasmid
  • transform into yeast
  • patch plate transformants onto tributryin plates plus methanol
  • observe halos
  • PCR to check integration
45
Q

How can a 1kb DNA ladder allow calc of amount in each DNA band on gel?

A
  • 5µl DNA ladder loaded onto gel corresponds to 0.5µg total DNA
  • if loaded 10µl, amounts of DNA for each band would have to be doubled to calc amount for each
46
Q

What must you be aware of when running a gel for a long time w/ a 100bp ladder?

A
  • smallest bands can migrate off end of gel

- so when counting bands, best to count from top or identify stronger bands w/in ladder

47
Q

How do you ensure DNA is clean and that vector is ready to receive the insert?

A
  • cut vector treated w/ enz to prevent re-ligation of 2 ends

- both products must be purified in same way

48
Q

Why is it important to quantify the amount of DNA present in purified samples of fragments?

A
  • as success of DNA ligation reactions dep on approp ratio of insert and vector DNA being present
49
Q

How do use a ladder to assess conc of DNA in sample?

A
  • compare brightness and thickness of bands (not bp size)
  • this gives you total amount of DNA in band, in ng
  • convert this to conc, using vol of DNA loaded
50
Q

What is an alt method for quantifying conc of DNA in samples, and how does it work?

A
  • nanodrop
  • by measuring absorbance
  • works on tiny vols (1/2µl)
51
Q

What controls were used when transforming ligation into competent E. coli?

A
  • ve
  • cut unligated vector = to give indication of how much uncut/self ligated vector in ligation reaction
  • water = show if any contamination or something wrong w/ ampicillin

+ve
- each vector to determine transformation efficiency

52
Q

What are the functions of the loading buffer in gel electrophoresis?

A
  • makes sample denser so sinks to bottom of well
  • allows us to see whats happening to the sample as its loaded
  • allows us to track progress of gel fractionation, checking it is running in right direction and seeing how far separation has proceeded
53
Q

Why did we run an aliquot of pJJ282 vector after digestion, before storing rest in the freezer?

A
  • to check digest had worked
54
Q

Why is it critical not to add loading buffer to whole of pJJ282 digest (just to sample running)?

A
  • loading buffer will inhibit action of alkaline phosphatase

- so would no longer be able to prevent self ligation

55
Q

Why did we run a sample of undigested pJJ282 vector on gel alongside digested vector?

A
  • need control to compare w/ digested sample
56
Q

Why did we add all of pHGL3c digest to prep gel?

A
  • isolate expression cassette from gel after fractionated away from plasmid vector
57
Q

What was the purpose of the 1hr incubation at 37° before plating transformations?

A
  • allows bacteria to recover from heat shock
  • also allows time for expression of antibiotic resistance gene before plating on ampicillin
  • skipping this step would make transformation step minimal
58
Q

What is the role of an enzyme master mix?

A
  • useful for saving time and reducing enzyme wastage

- also ensure that each reaction receives an identical conc of each component

59
Q

Why should you avoid vigorous mixing of sample during alkaline lysis?

A
  • would shear chromosomal DNA and lower plasmid purity
60
Q

In the plasmid miniprep what was the white ppt formed during the neutralisation step made up of?

A
  • ss gDNA, SDS and denatured cellular proteins, stuck together through hydrophobic interactions
61
Q

Why was DNA linearised before transformation in H. polymorpha?

A
  • only way DNA can be maintained in cell after transformation is integrates into chromosome
  • as no yeast origin or rep in pHGL10(R)
  • if plasmid DNA linearised w/in seq also present on chromosome, homologous recomb targeted between plasmid ends and results in stable integration
62
Q

Why did we expect only yeast cells that had received pHGL10(R) DNA to be able to form colonies?

A
  • A16 host strain carries leu2 mutation and cannot grow unless medium contains leucine
  • pHGL10(R) carry WT leu2 gene and would allow transformant to grow on synthetic medium lacking leucine
63
Q

How should growth on 2 plates compare when patch yeast colonies onto plate w/ methanol and plate w/ glucose?

A
  • similar level of growth on both
64
Q

Why did we patch out our yeast colonies onto tributyrin?

A
  • acts to induce gastric lipase expression
65
Q

What is the basic checklist for designing an experiment?

A
  • decide area to be tested
  • formulate hypothesis
  • decide which dependent variable to measure and how
  • plan statistical analysis of results

MBB265 (3 decks)

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