Transcript Rate of things - spectroscopy POST

Rate of things via
spectrophotometry
Aph 162, Winter 2009
Week 2
Overview
• 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 today
Spectrophotometry:
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 spectrum
Spectrophotometer
Spectrophotometer
The 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)
z
σ
The 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
σ
-σ(λ)·c·
l
-ε(λ)·c·
l
I1 (λ) = I0e
= I010
z
The 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
σ
-σ(λ)·c·
l
-ε(λ)·c·
l
I1 (λ) = I0e
= I010
• Absorbance=A(λ)= -log(I1/I0)=ε·c·l
z
The 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
σ
-σ(λ)·c·
l
-ε(λ)·c·
l
I1 (λ) = I0e
= I010
• Absorbance=A(λ)= -log(I1/I0)=ε·c·l
• ODλ=600=A/l = ε(λ=600nm)·c ~ c
• Units of OD: per unit length
z
When the law is applicable
Calibration –
measuring background
• Always need to measure “blank” - just medium.
• The spectrophotometer subtracts this measurement
from the actual measurement
A 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. cfu
How to do it in the lab
Plate every 30min
Try DX10 and D/10 as well
Next day:
http://micro.fhw.oka-pu.ac.jp/lecture/exp/images/cfu-7.jpg
Bacterial growth curves –
single carbon source
Growth 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 medium
Growth 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 stress
Bacterial growth curves –two carbon source:
catabolite repression
Catabolism: biochemical reaction leading to production of usable energy
How does it work?
CAP activator
LacI repressor
(constitutive)
cAMP Allolactose
glucose lactose
CAP =
catabolite
activator
protein
High glucose: Catabolite repression
High
OFF
glucose
When glucose is present → no activator → this
operon as well as operons for other sugars are shut
off.
Low glucose: Lactose switch
High
OFF
glucose
Lactose:
Low
High = ON
glucose
Low = OFF
When 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 growth
Experiments 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 time
Homework
• 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? Why?