Rate of things via spectrophotometryOverviewSpectrophotometry: The Beer-Lambert lawSpectrophotometerSlide 5The Beer-Lambert lawSlide 7Slide 8Slide 9Slide 10Calibration – measuring backgroundA standard curveA standard curve (cont.)How to do it in the labBacterial growth curves – single carbon sourceGrowth phasesSlide 18Slide 19Bacterial growth curves –two carbon source: catabolite repressionHow does it work?High glucose: Catabolite repressionLow glucose: Lactose switchWhen will the diauxic shift occur?Experiments for todayHomeworkRate of things viaspectrophotometryAph 162, Winter 2009Week 2Overview•Spectrophotometry–The Beer-Lambert law –Some weird units: OD600 and cfu’s–Calibration: a standard curve (OD600 vs. cfu)•Bacterial growth curves–Growth on a single carbon source–Growth on a two carbon sources (diauxic growth/catabolite repression)•Experiments for todaySpectrophotometry: The Beer-Lambert law •Relates concentration to the optical measurement of ‘absorbance’–Example: E. coli concentration• Combined with spectrophotometry can be used to distinguish and compare different molecules in solution–Example: Chlorophyll spectrumSpectrophotometerSpectrophotometerzσThe Beer-Lambert law•I0 = incident light (W/cm^2)•c = Number density of absorbers (e.g. cells)•σ(λ) = particle cross section (cm^2)•l = width of cuvette (usually 1cm)zσThe Beer-Lambert law•I0 = incident light (W/cm^2)•c = Number density of absorbers (e.g. cells)•σ(λ) = particle cross section (cm^2)•l = width of cuvette (usually 1cm) •For dilute samples: dIz/Iz =-σ·c·dz I1 (λ) = I0e-σ(λ)·c·l= I010-ε(λ)·c·lzσThe Beer-Lambert law•I0 = incident light (W/cm^2)•c = Number density of absorbers (e.g. cells)•σ(λ) = particle cross section (cm^2)•l = width of cuvette (usually 1cm) •For dilute samples: dIz/Iz =-σ·c·dz I1 (λ) = I0e-σ(λ)·c·l= I010-ε(λ)·c·l•Absorbance=A(λ)= -log(I1/I0)=ε·c·lzσThe Beer-Lambert law•I0 = incident light (W/cm^2)•c = Number density of absorbers (e.g. cells)•σ(λ) = particle cross section (cm^2)•l = width of cuvette (usually 1cm) •For dilute samples: dIz/Iz =-σ·c·dz I1 (λ) = I0e-σ(λ)·c·l= I010-ε(λ)·c·l•Absorbance=A(λ)= -log(I1/I0)=ε·c·l •ODλ=600=A/l = ε(λ=600nm)·c ~ c•Units of OD: per unit lengthWhen the law is applicableCalibration – measuring background•Always need to measure “blank” - just medium.•The spectrophotometer subtracts this measurement from the actual measurementA standard curve•OD600 doesn’t give absolute cell concentration•OD600 is cell dependent•Need to independently measure cell concentration so that the two can be related. This is called a standard curve.A standard curve (cont.)•Measure absolute cell concentration by dilution and plating. •Plating measures cfus = colony forming units•Standard curve = plot OD600 vs. cfuHow to do it in the labhttp://micro.fhw.oka-pu.ac.jp/lecture/exp/images/cfu-7.jpgPlate every 30minNext day:Try DX10 and D/10 as wellBacterial growth curves – single carbon sourceGrowth phases•Lag phase–Occurs upon inoculation–Duration depends on history of inoculum (exponential/stationary/damaged/type of medium)Growth phases•Exponential phase–Healthy cells –Cell number increases exponentially with a well defined doubling time–Reproducible physiological state–OD600 ~ 0.1 –Doubling times can be 20mim, hours, weeks and even months depending on the organism and growth mediumGrowth phases•Stationary phase–Population reaches steady state because•An essential nutrient becomes limiting•A waste product generated by the culture inhibits further growth–Physiological state of cell completely changes: cells are in stressBacterial growth curves –two carbon source: catabolite repressionCatabolism: biochemical reaction leading to production of usable energyHow does it work?CAP activator (constitutive)cAMPglucoseLacI repressorAllolactoselactoseCAP = catabolite activator proteinHigh glucose: Catabolite repressionHigh glucoseOFFWhen glucose is present → no activator → this operon as well as operons for other sugars are shut off.Low glucose: Lactose switchHigh glucoseOFFLow glucoseHigh = ONLactose:Low = OFFWhen will the diauxic shift occur? Experimental setup:•1L of glucose at 0.1g/L•Inoculums at t=0 is 10mL of saturated E. coli culture (@ OD600 = 1.5)•Rich medium (with casamino acids)•Doubling time: 20 min•Aerobic growthExperiments for today•Choose a growth medium–Glucose+Lactose/Matlose/Sorbitol (1:1 ratio, 0.1 g/L)•Measure OD600 every 5-10min (esp. near shift)–Don’t forget to blank before each measurement!–Minimize time incubator is open•Shift should occur at OD~0.25•Every ~30 min plate cells–Remember: OD600=1 ↔ 109 cells/mL• Note absolute timeHomework•Plot growth curve on a log scale–Identify all growth phases–Analyze your results in light of our discussion on catabolite repression•Extract doubling times by linear regression–Do your values make sense?•Plot standard curve (OD600 vs. cell count)–Is it linear? Are there errors?