Bacterial Production Lab

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Transcript Bacterial Production Lab

Bacterial Production Lab
State variables and processes
G: Bacterial Growth Rate (gC h-1)
DOM
State Var.
U
Other compounds
(e.g., EtOH)
B
Process
CO2
Objective: Measure bacterial growth rate (also called bacteria production)
Why do we want to measure processes?
Turnover: [B]/G
DOM
B
DIN
Z
Concentration
B
Z
DOM
DIN
Time
Growth Equations
td
Where:
x (t )  x (t0 )2
t t0
td
Doubling time of population.
Number or mass of cells per unit volume at time t.
td
x(t)
Note, cell mass or numbers are easily converted if we
assume cells are all the same size: x(t) = n(t), where  is
the mass per cell and n is the number of cells per unit
volume and x(t) is the mass of cells per unit volume.
Specific growth rate, 
Take derivative of above equation with respect to time.
Specific growth rate
dx(t )
 x(t )
dt

ln( 2)
;
td
dx
t


dt

x
(
t
)

x
(
0
)
e
x 
da f (t )
df (t )
 a f (t ) ln( a)
Recall:
dt
dt

1 dx (t )
x (t ) dt
Doubling rate
2 
1
; 2  
td
How are growth rates measured?
Accumulation or Loss Rates
G
O2
Isolate bacteria (How?), then measure:
G
B
CO2
dx(t ) dCO2
dO

 2
dt
dt
dt
What is main problem with this technique?
Use a Tracer
DOM
B
G
Tracer Requirements
Z
• Should not change environment
• Not preferentially consumed.
CO2
GB 
[ Blue (t )]  [ Blue (0)]
G
; G B
t
f
where GB is the rate of “blue” accumulation
and f is the fraction of DOM that is labeled
“blue”.
• Bacteria must utilize for growth
• Must be able to measure at low
concentrations. Low detection
limits reduce incubation times.
• Need some measure of f
Radio-isotope Tracers
Radionuclides typically used in biology:
Half Life
Type
Tritium (3H)
12.26 y

Carbon-14 (14C)
5730 y

35
Sulfur-35 ( S)
87.2 d

36
Chlorine-36 ( Cl)
300,000 y

Phosphorus-32 (32P)
14.3 d

Iodine-131 (131I)
8.06 d
, 
Iodine-125 (125I)
60 d

Types



For bacterial production,
3H and 14C used.
Note, 3H and 14C are weak 
emitters, so shielding is not
required.
Units: Curie, Ci:
Becquerel:
2.2  1012 disintegrations per min (DPM)
1 DPS = 60 DPM
Specific activity (SA):
Concentration:
Ci mmol-1
Ci ml-1
Radioactivity measurements:
• Geiger counter
• Scintillation counter (method we will use)
Measurements are given in counts per min. (CPM)
Due to some losses, CPM < DPM
Helium nuclei
Electron
Gamma ray
Levels of detection
SA: 371 mCi (mmol
14C)-1
Measure: 10 CPM~10 DPM
Conc:
1  10-14 mol
10 fmol
Annual Limit on 14C Ingestion: 2 mCi
Radiation Exposure Limits and Comparisons (UC Davis)
Dose Equivalent Limits (Monitored Radiation Workers)
Targe Tissue
Regulatory Limit
UC Davis Guideline
Whole Body
5000 mrem/year
2500 mrem/year
Extremities
50000 mrem/year
25000 mrem/year
Skin of the Whole body
50000 mrem/year
25000 mrem/year
500 mrem/gestational period
50 mrem/month
Fetus
Common Radiation Exposures (Natural Sources and Human Made)
One Coast to Coast Flight
Natural Background Radiation in the U.S.
3 mrem
150 - 300 mrem/year
Chest Radiograph, A/P view
15 - 25 mrem/view
Chest Radiograph, Lateral view
50 - 65 mrem/view
Screening Mammography (film/screen combination)
60 - 135 mrem/view
Computerized Tomography of Body (20 slices)
3000 - 6000 mrem
Biologically Significant Radiation Exposures (Absorbed/Acute Exposure)
Risk of contracting cancer increased 0.09%
1000 mrem
Temporary blood count change
25000 mrem
Permanent sterilization in men
100000 mrem
Permanent sterilization in women
250000 mrem
Skin Erythema
300000 mrem
Cataract formation
600000 mrem
What Compounds to Label?
Can’t 14C-label all DOM, so label only certain compounds
Glucose?
1000 CMP
Glucose
6000 CPM
B
5000 CMP
CO2
Can measure growth efficiency if CO2 is captured.
What fraction of the bacterial cell is produced from glucose?
CO2
Glucose
DOM
B
CO2
Glucose
Starch
Glucose
B
B
Problem: it is difficult to know what fraction of bacterial synthesis comes from glucose.
Label macromolecules instead using appropriate monomer:
Protein
RNA
DNA
Monomer
Amino Acids
A, G, C, U
A, G, C, T
% CDW
55.0
20.5
Cultured E. coli
3.1
Bacterial Production from 14C-Leucine Uptake
Use 14C-leucine to measure the rate of bacterial protein synthesis. Calculate
bacterial production rate using the following “pseudo constants”:
Pseudo constants:
Leucine content in protein
Protein Ave MW
Protein
Cell dry weight (CDW)
7.3 mol %
131.9
63 % CDW
54 % Carbon
Isotope Dilution Problem
Occurs when radioisotope is mixed with nonradioisotope.
Glucose
• Extracellular
• Caused by presence of Leu in solution.
• Leu Concentration is small (< 1 nM), so add
>10 nM Leu and ignore extracellular dilution.
LeuI
• Intracellular
• Caused by de novo Leu synthesis.
• Assume negligible, or measure.
LeuExt
Biosyn.
Assessing Isotope Dilution
Extracellular dilution:
• Measure background leucine concentration.
• Construct kinetic curve (top right fig).
• Construct time course curve (bottom left fig).
Uptake Rate
1
0.8
0.6
0.4
0.2
0
0
Intracellular dilution:
20
40
60
80
100
Leu Conc. (nM)
• Measure actual protein synthesis rate and
compare isotope-measured value.
• Often, intracellular dilution is assumed not to
be significant.
Leucine Incorporated
• Measure Sp. activity of Leu in protein.
2
1.5
1
0.5
0
0
20
40
60
Incubation time (min)
80
Example Calculations
700000
y = 20657x - 9750
600000
500000
DPM
Experimental Setup
SA Leu:
100 Ci mmol-1
Incubations:
15 and 30 min
Volume
1.5 ml
400000
300000
200000
Measure activity after incubation
20,657 DPM (from slope)
Note, CPM ≠ DPM
100000
0
0
10
20
30
40
Time (min)
DPM
1
Ci
1 mmol Leu
1 mol Leu



12
min 2.2  10 DPM 100
Ci
1000 mmol Leu
mol Leu
 9.39  10 14
min
mol Leu
1 mol Protein
g Protein
1 g DCW
gC
Cells  9.39  10 14

 132

 0.54
min
0.073 mol Leu
mol Protein 0.63 g Protein
g DCW
gC
 1.46  10 10
min
gC
min
1
ml
gC
BP  1.46  10 10
 1440

 1000
 1.40  10 4
min
d 1.5 ml
l
ld
gC
gC 1 mol C
mol C
 1.40  10 4
 10 6

 11.7
ld
gC 12 gC
ld
Leu  20657
Notes
• Similar procedure can be done using thymidine incorporation into DNA.
• Centrifugation plus rinsing (or filtration plus washing) is used to separate
added Leu from bacterial incorporated Leu.
• A killed control is run under identical conditions to account for abiotic
adsorption of Leu onto particulate matter.
• Isotope dilution due to extracellular matrix may not be insignificant in
eutrophic environments.
• Conversion factors are dependent on cellular conditions, and values
reported are controversial. Often, only Leu incorporation is reported
(i.e., not converted into cell biomass).
Sea Level Rise
Land Use Change
Plum Island Estuary
Example: Isotope Dilution
Byron Crump
Leucine saturation curves
4500
17-Jul-00
leucine incorporation (pmol/l*h)
4000
25-Jul-00
3500
3-Aug-00
3000
2500
2000
1500
1000
500
0
0.00
50.00
100.00
150.00
added 3H-leucine (nM)
200.00
Example: Plum Island Estuary Survey
(Byron Crump)
Bacterial activity
4000
July 11-12, 2000
Leucine incorporation (pmol/l*h)
3500
3000
2500
2000
1500
1000
Whole water
free-living (<1 um nitex)
500
0
0
5
10
15
20
25
30
Conductivity
35
40
45
50