Lecture 6: Bacterial Growth

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Lecture 6 Bacterial Growth
Reading assignments in Text Lengeler et al.
1999 Text pages 88-90, 95-103, 108-109 Growth
and nutrition Text pages 541-544 E. coli
symmetric division Text pages 571-574
Caulobacter asymmetric cell division Text pages
882-884 Making MSG Lecture 5 Text pages
205-214, 229-232 Examples of organotrophy Text
pages 234-244, 267-268 Lithotrophs in
general Text pages 245-259, 911-12 Lithotroph
specifics Text pages 327-340 Phototrophs Text
pages 67 Photophosphorylation Lecture 4 Text
pages 116-122 Assimilation Text pages 177-182
Assimilation reactions Text pages 155-157
Storage compounds
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Last lecture
(get their energy)
All organisms
Fix CO2
Assimilate C
?
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Lecture Overview
Bacterial populations (lab conditions)
Food/ Media
Sterilization
Growth measurement
Changes during growth
Applications Making MSG
Bacteria as single cells (cell cycles)
E. coli divides symmetrically
Caulobacter crescentus divides asymmetrically
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Food/ Media
undefined from organisms
defined from chemicals
E. coli LB (Luria Bertani)
M9
per liter water
5 g Yeast Extract
3 g KH2 PO4
10 g NaCl
0.5 g NaCl
(15 g Agar for plates)
1 g NH4Cl
Autoclave
(steam pressure)
MgSO4 1 mM
CaCl2 0.1 mM
The truth is seldom simple, but never
pure. -Oscar Wilde
?
Done ?
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Sterilization
Why sterilize?
What is Pasteurization?
Methods
Autoclaving
Filtration
Radiation
Chemicals
?
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Bacterial growth and measurement
Exponential growth
Log growth
DT Doubling Time
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Measuring N
1 Viable cell counting
Sample
Dilute (note dilution factor)
One cell One colony 106 cells
2 Direct counts
side
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Measuring N
3 Automated flow cell/ cytometry
Volume (flow) x (time)
4 Absorbency or Optical Density (OD)
OD logI0/I(cells)
OD log10/1
1.0
109 cells / ml
I (cells)
Useful linear range OD 0.1 to 1.0
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Changes during growth
?
1 Induce survival genes
ss
2 Fewer ribosomes
3 Smaller cells (gt8-fold range)
4 Modified fatty acids in P-lipids
5 High mutation rates in sub-population (?)
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Making Mono-Sodium Glutamate (MSG)
(see Course Pack lecture 6, Text pages 882-4)
800,000 tons / year
What is MSG ?
Industrial production principles
Central metabolism
Food Glucose Ammonium Salts
MSG (secreted)
?
Synthetic PW Glutamate Dehydrogenase (GD)
Biotin
Growth control ?
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Making MSG from Central Metabolism
Growing bacteria Biotin
Cells MSG Factories
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Single cell growth Cell division cycles
Continuous processes
Most biosynthesis (including cell wall)
Ribosome assembly
Punctuated events
Chromosome replication
Cell/wall division
Chromosome Replication
1 Commitment (?)
Critical cell mass trigger
2 Start (assemble) replication factories
Timing (?)
Place oriC
Assemble replication factories (?)
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E. coli Chromosome replication (continued)
3a DNA synthesis
2 x 2x106 bp
3b Polar orientation of oriC and chromosome
movement (?)
4 Termination
6 ter sites pause sites
balance replication
Mechanism
5 Resolution and separation
Gyrase Topo IV unlink
Topological DNA linkage
Covalent linkage problems
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5 Resolution and separation
Homologous recombination (RecA, BCD)
Even X-over
Steps 1-5 C period 40 min best time
Paradox ?
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Site-specific DNA resolution
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E. coli cell division
1 Commitment (?)
Chromosome-free mini-cell
FtsZ mono-mer (GTP) tubulin-like protein
PBP3
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Staggered projects 2, 4, 8, 16 chromosomes inside
one large cell
Feast or famine strategy
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Caulobacter crescentus
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Asymmetric strategies
Stays and makes more Swarmers
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Cell cycle with swarmer differentiation
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Symmetric and Asymmetric protein distribution
C. crescentus early division
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Caulobacter applications
Fish farming
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Michaelis-Menton -like analysis of growth
R growth rate
0
C Conc. of limiting nutrient
Where on the curve ?
In batch cultures ?
In a turbido-stat ? a chemo-stat ?
In nature ?
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Controlled growth conditions
Batch cultures or Fermentation cultures
Continuous cultures
Turbido-stat
C gtgt Km
Dilution rate must growth rate (needs extra
feedback control)
Chemo-stat
C Km, C lt Km
Cell conc. constant x C
Growth rate R Media flow rate
(i.e. adjust Y-axis and slope)
Reflect natural growth-limiting environments
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