Functional Genomics: Progress with Physiology and Genetic Tools

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Functional Genomics Progress with Physiology and
Genetic Tools
Judy D. Wall Kelly Bender Brett Emo Joseph A.
Ringbauer, Jr. Huei-Che Bill Yen Grant Zane
Biochemistry Department University of
Missouri-Columbia
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Outline of Progress

• Physiology Cost of growth with sulfate
• Basic genetic tools
• Useful antibiotic resistances
• Electrotransformation
• Conjugation
• Mutant isolation
• Transposon mutagenesis
• Plasmid insertion mutagenesis
• Marker exchange mutagenesis
• Tagging proteins
• Controlled expression of proteins

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Physiology Cost of growth with sulfate
PPi
ATP
AMP
S2O3
End product
Sulfate costs 2 ATPs more than sulfite as
electron dump, but allows eight versus six
electrons to be removed.
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Relative Yields of Electron Acceptors
Protein
77 µg/ml 222 µg/ml 62 µg/ml
Growth with sulfate is a stress on these bacteria
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Optical densities can fool you!
10 µg/ml
12 µg/ml
93 µg/ml
132 µg/ml
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Useful antibiotic resistances
For selection of gene transfer Carbenicillin 25-5
0 µg/ml Chloramphenicol 30-40 µg/ml Kanamycin 7-
800 µg/ml G418 400 µg/ml For
counterselection, spontaneous mutants of D.
vulgaris resistant to Nalidixic Acid 200
µg/ml Rifampicin 400 µg/ml
Most used!
Sodium ion concentrations have a dramatic effect
on effectiveness of some antibiotics and
inhibitors, e.g., Kanamycin
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Electroporation Procedure
1750 V
Dilute to 1 ml, transfer, and recover over night
in the anaerobic chamber at 37ºC.
50 ml culture to 1 ml in 30 mM PIPES 1 µg plasmid
DNA
Plate with antibiotic selection.
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O.D. vs. Transformation Efficiency
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Transposon mutagenesis
Generous gift of Bill Metcalf
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Location of Transposon Insertions in D. vulgaris
Chromosome
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Example of Transposon Insertion Site
uvrBminiTn5
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Phage Shock Protein A
Kanr
Ampr

• In E. coli, helps maintain proton motive force.
• Induced under stress conditions.
• Essential for stationary phase survival
  under alkaline pH.

Note pspA in D. vulgaris was responsive to oxygen
exposure and increased in stationary phase!
pMO705
colE1 ori
f1 ori
socE
LacZ
pspA
-3
79
166
pcpB
pspC
pspF
pspA
Integration of plasmid into chromosome
PspA-22F
socE
Kanr
pspF
colE ori
pspA
523 bp
Ampr
LacZ
F1 ori
pspA
pcpB
pspC
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Deletion Construction
Protocol for mutagenic vector construction
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Mutagenic Cassette
Clone into delivery plasmid
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Recombination Event 1 The first recombination
event integrates the vector and forms an
intermediate product that is ampicillin and
kanamycin resistant and sucrose sensitive.
Recombination Event 2 The second recombination
event removes all vector sequence, the target
gene, and the plasmid component of the junctions
leaving an ampicillin sensitive, kanamycin and
sucrose resistant deletion mutation.
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Vector sequence D.vulgaris sequence
AmpicillinR cassette sacB sequence
26 base common sequence 20 base unique
barcode KanamycinR cassette
Target gene sequence
ab
cd
/
?
a b
c d
Recombination 1
a b
cd
/
c d
?
a b
Recombination 2
cd
a b
/
?
c d
ab
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Outline of Progress

• Physiology Cost of growth with sulfate
• Basic genetic tools
• Useful antibiotic resistances
• Electrotransformation
• Conjugation
• Mutant isolation
• Transposon mutagenesis
• Plasmid insertion mutagenesis
• Marker exchange mutagenesis
• Tagging proteins
• Controlled expression of proteins

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Near Future Objectives

• Enrich for specific phenotypes among transposon
  mutants
• Provide transposon mutants for Hazen group
  analysis
• Generate deletions from mutagenic cassettes for
  further analysis by all groups
• Prepare tagged protein constructs from cloned
  genes from Diversa
• Confirm presence of regulatory small RNAs in D.
  vulgaris and provide mutants of these elements
  for mircroarray analysis
• Establish the species designation of D.
  desulfuricans G20 in collaboration with Eric Alm

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1 Day
7-14 Days
3 - 5 Days
No
No
Choose Marker
Obtain Sequence
Design Primers
Order Primers
PCR1
Repeat PCR1
R1
D1
Yes
Yes
No
Barcode
Common
Store
D2
Yes
Choose Gene for Deletion
Obtain Up/Down- Stream Seq.
Design Primers
Order Primers
PCR2 and PCR3
Gel Purify
R 1. Construct mutagenic plasmid R 2. Construct
mutant
No
Yes
No
PCR4
Repeat PCR2 and PCR3
D3
Yes
D1. Did PCR1 work? D2. Are products 2 and3
ready? D3. Did PCR2 and PCR3 work? D4. Did PCR4
work? Post on Biofiles Minimum times
are indicated.
No
No
No
Repeat PCR4
Change PCR Conditions
Close Book
D4
Yes
Yes
Gel Purify
B
Ligate into Cloning Vector
Ligate into Mut. Vector
R2
B
B
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R2
5-7 Days
Electrotransformation into SRB
Conjugation into SRB
Yes
Purify Ab1R CFU
Purify Ab1R CFU
7 Days
Yes
D5
No
Confirm Plasmid Mutation
R 2. Construct mutant First Recombination for
Plasmid Integration R 3. Construct mutant
Second Recombination
1-3 Days
Post to Biofiles Minimum times are
indicated. D5. Is there E. coli
contamination? D6. Is plasmid integrated as
expected?
PCR Junction Product
Southern of Digested Chromosome
B
Yes
No
No
D6
R3
R2
Close Book
B
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R3
Enrich SucroseR
R 3. Construct mutant Second Recombination R4.
Characterize Mutant
7 Days
Plate SucroseR and Ab1R
Post to Biofiles Minimum times are
indicated. D7. Is plasmid integrated as expected?
B
Screen Plasmid Ab2S
Confirm Plasmid Mutation
1-3 Days
PCR Junction Product
Southern of Digested Chromosome
Yes
No
7-30 Days
No
D7
B
3 Days
Growth Curves Carbon sources Stress responses
R4
R3
Grow and Make Glycerol Stock
Ship to LBL for Biomics
Close Book
B
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Electro-transformation into D.vulgaris
Purify Ab1R CFU
R1
Yes
Choose Marker
Choose Gene for Deletion
No
Confirm Plasmid Mutation
Purify Ab1R CFU
R2
Conjugation into SRB
D5
B
Obtain Up/Down- Stream Seq.
Obtain Sequence
1 day
Barcode
Common
PCR Junction Product
Southern of Digested Chromosome
No
Design Primers
Design Primers
B
D6
Yes
No
Order Primers
Order Primers
7-14 days
B
R3
Close Book
R2
PCR1
PCR2 and PCR3
R 1. Construct mutagenic plasmid R 2. Construct
mutant
R1
Enrich SucroseR
R2
No
No
Repeat PCR
D1
D3
Yes
Yes
Did PCR1 work? Are products 2 and 3 ready? Did
PCR2 and PCR3 work? Did PCR4 work? Post to
Biofiles
D1
Plate SucroseR and Ab1R
D2
1-2 days
No
Store at 20oC
D3
D2
Screen Plasmid Ab2S
D4
B
Gel Purify
Gel Purify
Confirm Deletion Mutation
PCR4
R2
Construct mutant First Recombination for Plasmid
Integration Construct mutant Second
Recombination Characterize Mutant
Close Book
PCR Junction Product
Southern of Digested Chromosome
R3
No
R4
Repeat PCR4
Change PCR Conditions
D4
Yes
Yes
No
D7
D5
Is there E. coli contamination? Is plasmid
integrated as expected? Is plasmid integrated as
expected? Post to Biofiles
Yes
Gel Purify
D6
R3
Grow and Make Glycerol Stock
Close Book
D7
2 days
B
B
Ligate into Cloning Vector
R4
B
Ligate into Mut. Vector
R2
B
Growth Curves Carbon sources Stress responses
Ship to LBL for Biomics
B
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Basal Medium without Carbon or TEA
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Physiology Cost of growth with sulfate
Blue substrate level phosphorylation possible
Brown end products