Flashcards in Semester 1 Deck (66):
Typically how long are covalent bonds in proteins, and what makes these distances shorter?
- C-C ≈ 1.5Å
- shorter if double/triple bond character involved
Typically how long are hydrogen bonds in biological systems?
Where in protein can hydrogen bonds be formed?
- 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
Hos is it poss for diff protein chains to adopt distinct conformations and shapes?
- polypeptide backbone possesses inherent flexibility, as rotations poss about certain bonds in main chain
What 2 angles in the main chain can vary?
- phi = rotation around bond joining α C to peptide N
- psi = rotation around bond joining α C to carbonyl C
What 3 groups can conformation of polypeptide at each residue be classified into?
- 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
When might 'disallowed' conformations be allowed?
- some exceptions where other favourable interactions offset energy penalty paid for being in disallowed region, eg. specific functional role
What is the polarity of the polypeptide chain?
- N --> C ter
Why are 3^10 helices less energetically stable as α-helices?
tighter helix so...
- non ideal H-bonds
- pot sidechain clashes
- loss of internal packing
- less favourable phi and psi angles
Is Serratia gram +ve or -ve?
Why is Serratia a good model for opportunistic pathogens?
- (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
What are pathogenic Serratia species almost always assoc w/?
- nosocomial (hospital acquired) infections
- or intravenous drug users
What is the key feature which makes Serratia adaptable as an opportunistic pathogen?
- 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
What is the role of prodigiosin (Pig) in Serratia?
- antibiotic w/ antibacterial, antifungal and antiprotozoal activities
- 2° metabolite --> no function in cell growth
Where is Pig found in Serratia, and why?
- hydrophobic, so tends to remain assoc w/ cell membrane
Apart from Pig, what other 2° metabolite does Serratia prod, and how does it differ?
- 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
How may Pig and Car allow Serratia to compete better?
- killing off other non-Serratia bacteria in env
What are the most important virulence factors made by Serratia?
- damaging secreted enzymes, inc proteases and DNAses, which degrade host tissue macromolecules, releasing nutrients to allow growth
How can some species of Serratia also be pathogenic to plants?
- prod pectinases and cellulases that break down plant cell wall
When are all antibiotics and extracellular enzymes of Serratia made in large quantities?
- stationary phase, when cell density reached high levels
What is Quorum sensing, and how does it work?
- 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
Why does Serratia delay the synthesis of virulence factors until high no. of cells?
- 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
What are the 2 key components of quorum sensing in Serratia?
- enzyme that makes AHL
- regulatory binding protein, to enhance transcrip of target genes when AHL bound
What are 2 examples of systems under the control of quorum sensing in Serratia?
- pigment prod
- swarming behaviour
How can you demonstrate quorum sensing in Serratia on a streak plate?
- 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
What is a pleiotropic mutation, and what can they indicate?
- mutations affecting 2 or more phenotypes
- indicate regulatory connections
How was Serratia mutagenised for our mutant screens?
- transposon mutagenesis
How could you design an indicator plate for detection of a new phenotype in Serratia?
- 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
What can a cross feeding experiment tell you?
- order of action of genes in a pathway
What is cross feeding?
- 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
How does cross feeding show the order of genes?
- if mutant cross feeds another mutant
- gene defined by mutation in "feeder" must act later in pathway than mutant being "fed"
How did Serratia show cross feeding?
- D --> C
- D --> B
- B --> C
- so pathway is D --> B --> C
In a cross feeding experiment what acts as the control?
- middle part of streak not near another mutant
What would a pleiotropic mutant look like if defective for pigment, carbapenem and AHL production in Serratia?
- white (lacks pigment)
- lack purple halo on AHL plate
- lacks halo of ESS (carbapenem) plate
When might cross feeding not work?
- 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
What was the nature of the Serratia LIS mutant, and how do we know this?
- prevents prod, but not detection, of AHL
- can detect it as cross feeding from WT to LIS
- but cannot prod it itself
If LIS mutant is colourless, what does this suggest about control of Pig synthesis in Serratia?
- LIS can't prod AHL, but when externally supplied colony turns red
- suggests AHL req for upreg of pigment prod
What is a poss mol basis for the pleiotropic Serratia mutants?
- 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
How can swarming behaviour of Serratia be demonstrated?
- place on low % agar and observe colony behaviour
What is swarming?
- flagella driven coord movement of pop of bacteria across solid surface
Why is it advantageous for opportunistic pathogens to swarm?
- for tissue invasion
- overcoming host defences
- secretion of higher levels of extracellular enzymes and toxins
How do hard and soft-agar swarmers differ?
- 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
Why is transposon mutagenesis unsuitable for isolation of mutants in essential genes, and what could be used instead?
- 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
What is an expression cassette?
- part of vector DNA used for cloning and transformation
What is the overall procedure we used for transformation experiment?
- 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
How can a 1kb DNA ladder allow calc of amount in each DNA band on gel?
- 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
What must you be aware of when running a gel for a long time w/ a 100bp ladder?
- smallest bands can migrate off end of gel
- so when counting bands, best to count from top or identify stronger bands w/in ladder
How do you ensure DNA is clean and that vector is ready to receive the insert?
- cut vector treated w/ enz to prevent re-ligation of 2 ends
- both products must be purified in same way
Why is it important to quantify the amount of DNA present in purified samples of fragments?
- as success of DNA ligation reactions dep on approp ratio of insert and vector DNA being present
How do use a ladder to assess conc of DNA in sample?
- 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
What is an alt method for quantifying conc of DNA in samples, and how does it work?
- by measuring absorbance
- works on tiny vols (1/2µl)
What controls were used when transforming ligation into competent E. coli?
- 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
- each vector to determine transformation efficiency
What are the functions of the loading buffer in gel electrophoresis?
- 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
Why did we run an aliquot of pJJ282 vector after digestion, before storing rest in the freezer?
- to check digest had worked
Why is it critical not to add loading buffer to whole of pJJ282 digest (just to sample running)?
- loading buffer will inhibit action of alkaline phosphatase
- so would no longer be able to prevent self ligation
Why did we run a sample of undigested pJJ282 vector on gel alongside digested vector?
- need control to compare w/ digested sample
Why did we add all of pHGL3c digest to prep gel?
- isolate expression cassette from gel after fractionated away from plasmid vector
What was the purpose of the 1hr incubation at 37° before plating transformations?
- 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
What is the role of an enzyme master mix?
- useful for saving time and reducing enzyme wastage
- also ensure that each reaction receives an identical conc of each component
Why should you avoid vigorous mixing of sample during alkaline lysis?
- would shear chromosomal DNA and lower plasmid purity
In the plasmid miniprep what was the white ppt formed during the neutralisation step made up of?
- ss gDNA, SDS and denatured cellular proteins, stuck together through hydrophobic interactions
Why was DNA linearised before transformation in H. polymorpha?
- 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
Why did we expect only yeast cells that had received pHGL10(R) DNA to be able to form colonies?
- 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
How should growth on 2 plates compare when patch yeast colonies onto plate w/ methanol and plate w/ glucose?
- similar level of growth on both
Why did we patch out our yeast colonies onto tributyrin?
- acts to induce gastric lipase expression