Making a Stress Book

1
Making a Stress Book

• Scientific Flow Charts for GenomicsGTL Project
  at
• Lawrence Berkeley National Laboratory

2
WEEK 1-2
WEEK 3-4
Choose Perturbation
Growth and MIC
R4
R2
R1
WEEK 5-6
WEEK 7
yes
Design Time/Dose Series (S-FTIR)
Execute Time Series
d1
R6
R7
R3
E2
no
No, try again
No
Close Book
d2
R5
Close Book
Yes
E1
Store Backup samples
Ship to ORNL
For all reports there is an automated email sent
to the teams. Red means a data upload has to
occur. CC does analysis at this point. R1-
Start Stress Book- Record Name, Date, R2- Add
description of condition (protocol) and rationale
to stress book. R3- Add description of
experiment, upload biolog/growth, morphology
data, etc. complete phenomics, add conclusions
from experiment, note person, date, R4- Add
description of why decision was made R5- Upload
of S-FTIR time series R6- Upload experimental
design R7- Add comments, upload culture data,
assign sample IDs, delivery point, date shipped
added to ORNL samples. R8- Annotate that
shipment is received. Comments on conditions,
time to analysis, who is doing analysis,
protocols. R9- Upload analysis data. Annotate
with comments and decision.
R8
RNA Analysis
WEEK 7-8
E3
R9
No
Close Book
d3
Yes
Stage 2
D1- Do we believe we are seeing an interesting
response? D2- Did the time series work? (QA/QC
verification) D3- Is the RNA Good? (QA/QC
verification)
WEEK 9
Note AEMC can start new growth,MIC, and time
series design analyses as soon monthly. Nitrate,
nitrite, and salt have already been completed for
growth and MIC, FITC will be completed in the
next 2 months. Setup for time series can be done
and RNA analysis done to verify appropriateness
of samples for the rest of the team. Stored
samples can prioritized for the most interesting
responses.
3
WEEK 21-22
WEEK 10-20
WEEK 8-10
Metabolite Quality Report
Significant Responders Report
Metabolomics
R19
R11
WEEK 23
Separate and Ship Samples
ICAT Proteome
R20
R12
Stage 2
Significant Responders Report
R9
Proteomic Consistency/ Qual.Report
Diversa Proteome
R13
R21
Consistency/ Conclusions Report
Sandia DIGE
GO Report
R14
R22
Phenomics Summary Report
SEM
R23
Comparative Stress Report
R15
TEM
Yes
EPS Report
Significant Responders Report
R24
R16
EPS
Back to stage 1
d4
Lipidomics Report
No
R25
PLFA
R17
Operon/Regulon Report
Microarray Quality Report
Transcriptome
R10
R18
For all reports there is an automated email sent
to the teams. Red means a data upload has to
occur. CC does analysis at this point. R9-One
shipment goes to Missouri and Washington for
enrichment (DNA Isolation, Primary Enrichment,
DNA Amplification) Add shipping date to all
other samples. R10-17 Add receiving dates to
samples. Upload Exp. Design, protocols, contacts,
predicted delivery time.. R18- Microarray data
and Ecogenomics Report D4 Is microarray data ok?
Receive OK from CC before proceeding with
remaining Genomics R19-R25 Upload data, add
comments, Quality estimates, successful yes or
no, requests for special analysis, etc. Automate
analyses delivered back to stress book.Note If
method fails, there maybe should be a procedure
for requesting a backup sample. Can FGC do
analyses from different exp concomittantly to
increase throughput
GO Report
4
Specific comments for tentative timelines Week
1-2 LBNL(Hazen lab) Conduct MIC and growth
curve assays for stress. Checkpoint (CC) Week 3
4 LBNL(Hazen lab) Determine via S-FTIR time
response. Obtain biomass for dose dependence /
time course for QC Checkpoint (CC) Week 5
LBNL (Hazen lab) Provide above biomass to ORNL
for MA analysis Next 2-3 weeks Prepare to
scale up experiments (Obtain feedback/help from
other FGC labs) - for week 7 Conduct other
related assays e.g. IR and PLFA assays Post
data on biofiles Week 6 7 ORNL Obtain MA data
for biomass from dose dependence / time course
Provide data to Computational core
group and post on Biofiles LBNL(CC) Assess
quality of data in general as well as identify
time points that provide most striking
data via MA analyses. Email all and / or post on
biofiles. QC of data. Posts analysis on
Biofile Checkpoint (CC) Week 8 LBNL(Hazen
lab) Prepare large scale biomass for FGC
(helped by Keasling and Singh lab). Distribute
Biomass to FGC and archive additional
samples Week 9-10 ORNL Conduct MA for their
aliquot of the large Biomass. Provide data to
computational core group Comment on any
problems with sample. - Checkpoint (CC)
5
Week 10-14 (five weeks) Diversa Conduct
Proteomics 3-4 weeks till final data Post
data on Biofiles LBNL (Keasling lab) conduct
ICAT Proteomics experiments sample prep and
run 1-2 weeks data analysis - 2 weeks Post
data on Biofiles Sandia Conduct DIGE
experiments 1-3 weeks for sample prep, labeling
2D and obtaining spots for MALDI 2 weeks for
MALDI MS Post data on Biofiles Parallel
efforts during Week 1 4 and 5-10 on
underdeveloped Methods Metabolomics (Keasling
lab) Protein-Protein Interactions
(Sandia) Carbohydrate Profiling (Hazen lab
Keasling lab) Week 15 All labs Data
analysis CC Data integration and
modeling. All labs Participate in a Data
discussion meeting to summarize findings What
was expected in the stress? Does our data
corroborate these? Trends observed Up and
Down Use of controls to eliminate growth related
changes Co-relating MA with proteomics
data Support from EPS, PLFA, IR and Metabolite
assays Emerging hypothesis pathways and
operons affected by or involved in
stress Implications of the data models for
stress response. Candidates for molecular
studies e.g. Mutations, protein-protein
interactions and Biochemical assays e.g.
enzymatic activity Week 16 Compile above as a
report/manuscript
6
Stressors
Stressors should be chosen for two reasons 1)
They naturally occur at the sites of interest, 2)
they are classical stressors that allow us to
compare responses in our organisms to better
understood systems. The stressors we wish to
pursue are listed below. Synergistic effects of
stressor with metals and radionuclides may be
more important then some of the single response
to some of the classical stressors.
7
Environmental Characterization and Sequencing
8
WEEK 1-3
WEEK 4-5
Choose Site
Site Data
R4
R2
R1
WEEK 5-6
WEEK 7
yes
Design Field Sampling Plan
Execute Field Sampling
d1
R6
R7
R3
no
Ship to LBNL
R5
Close Book
E1
E2
No, try again
No
Close Book
d2
Yes
For all reports there is an automated email sent
to the teams. Red means a data upload has to
occur. CC does analysis at this point. R1-
Start ECS Book- Record Name, Date, R2- Add
description of site and rationale to book. R3-
Add site characterization data contaminants,
physical, chemical, biological, hydrology,
geology and any conceptual models of the site,
include links to other sites, investigators,
etc. R4- Add description of why decision was
made R5- Preliminary data for Conceptual Model of
site R6- Upload Field Sampling Plan R7- Add
comments, upload sample characterization data,
assign sample IDs, delivery point, date shipped
from ORNL to LBNL and from LBNL to Diversa, MU,
and UW samples.
Store Backup samples
Stage 2
WEEK 8
D1- Is this site interesting and relevant to
stressors we are studying? D2- Are the samples of
good quality? (QA/QC verification)
Note AEMC can start new field sampling with
minimal changes in sampling plan when going to
the same site eliminating the first 5 weeks of
preparation. Stored samples can be prioritized
for the most interesting responses.
9
WEEK 14-15
WEEK 10-14
WEEK 8-9
Stimulated Activity Report
Community Structure Report
UW/MU Enrichments
R16
R9
WEEK 16
MU/ORNL F Probe/MA
R17
R10
Ship Samples
Stage 2
Functional Population Report
R8
Ecogenomics Report
Diversa Clone Libraries
R11
R18
Conceptual Biogeochemical Model Report
LBNL/JGI 16s Microarray
Functional Activity Report
R12
R19
Geochemical Report
Physical
R20
Comparative Stress Report
R13
Chemical
Biomass Report
Significant Stressors Report
R22
R14
Direct Counts
Lipidomics Report
R23
PLFA
R15
For all reports there is an automated email sent
to the teams. Red means a data upload has to
occur. CC does analysis at this point. R8- Add
shipping date to all other samples. R9-15 Add
receiving dates to samples. Upload Exp. Design,
protocols, contacts, predicted delivery time..
R16-R23 Upload data, add comments, Quality
estimates, successful yes or no, requests for
special analysis, etc. Automate analyses
delivered back to book.Note If method fails,
there maybe should be a procedure for requesting
a backup sample.
10
Specific comments for tentative timelines Week
1-3 LBNL(Hazen lab) With ORNL FRC or others
collect data from site to define best sampling
locations. Checkpoint (CC) Week 4
5 LBNL(Hazen lab) Design Field Sampling Plan
with approval from Diversa, MU, ORNL, and WU.
Send permits, forms etc. to ORNL for prior
sampling and shipping approvals. Checkpoint
(CC) Week 5 6 LBNL (Hazen lab) Execute Field
sampling by NABIR FRC per their schedule, samples
shipped to LBNL. From field analyses and
shipping times determine if the quality of the
samples is appropriate for further analysis and
shipping to other labs. Week 7-9 LBNL (Hazen
lab) Ship to MU, Diversa, and WU from LBNL, or
arrange for sample prep to be done at ORNL or
LBNL. Week 10-14 LBNL(Hazen lab)
Biogeochemical analyses, PLFA, biomass, 16s
Microarray, DNA recovery for sequencing and
data from Site (NABIR FRC). UW Enrichments for
populations of interest, ID, and probing (FACS,
etc) MU Enrichments for other functional groups
and probing. Diversa Large insert and small
insert clone libraries
11
Week 14-15 All labs Data analysis CC Data
integration and modeling. All labs Participate
in a Data discussion meeting to summarize
findings Co-relating enrichments, biomass,
ecogenomics, geochemical, lipidomics,
sequencing Support from Stress studies and
comparative genomics pipeline Emerging
hypothesis stressors, community
response Implications of the data models for
stress response. Candidates for comparative
studies e.g. sequencing, stress studies,
population interactions and ecogenomics Week
16 Modify Conceptual Model of Biogeochemistry Co
mpile above as a report/manuscript
12
Deletion Project
13
1 Day
7-14 Days
3 - 5 Days
No
No
Choose Marker
Obtain Sequence
Design Primers
Order Primers
PCR1
Repeat PCR1
R 1
D1
Yes
Yes
Barcode
Common
No
Store
D2
Yes
Choose Gene for Deletion
Obtain Up/Down- Stream Sequence
Design Primers
Order Primers
PCR2 and PCR3
Gel Purify
R 1. Construct mutagenic plasmid R 2. Construct
mutant
Yes
No
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
R 2
B
B
14
PCR1 has AbR marker for exchange, molecular
barcodes, and common sequence for monitoring
mutants PCR2 has sequence upstream of target
gene, ATG of target, and common sequences PCR3
has common sequences, stop codon of target gene,
and sequence downstream of target PCR4 has three
preceding products joined PCR2/1/3
PCR4
TAA
ATG
Ab1R
Common A
Common B
Barcode 1
Barcode 2
15
R 2
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
PCR Junction Product
Southern of Digested Chromosome
Yes
No
No
D6
R 3
R 2
Close Book
B
16
R 3
Enrich SucroseR
R 3. Construct mutant Second Recombination R4.
Characterize Mutant
7 Days
Plate SucroseR and Ab1R
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
R 4
R 3
Grow and Make Glycerol Stock
Ship to LBL for Biomics
Close Book
B
17
Gene deletion in D.vulgaris Stages in creating
knockout mutants
Methods development
Gene deletion experiment
Antibiotics
B
Design and develop suicide vector
B
Obtain TAGs from CC
Select genes

• Set up transformation protocols
• Conjugation
• Electroporation

Start point
B
B
1-2 weeks
Design and order primers
post on Biofiles
B
PCR
PCR
Protocols, Vectors developed / used, List of
genes chosen for knock out, TAGs used in design,
and Progress in construction vectors as well as
strains made are all useful if posted on Biofiles
2-3 weeks
Clone in suicide vector
B
gt 1 week
sequence
18
Transform E.coli donor - S17-1
1 week
B
1 week
Grow S17-1
Prepare LS4D plates and media
Grow Dv wt
Set up conjugation
1 - 2 weeks
Screen for colonies
1- 2 weeks
Stocks
grow
B
colony pcr
southern
biolog
2 - 4 weeks
B
B
19
Molecular Complexes
20

• Molecular Complexes
• Isolation Three Parallel Approaches
• Antibody pull down approach
• Tagged Protein approach
• Endogenous
• Exogenous

• Identification
• SDS-PAGE MALDI-MS
• Nano-LC QTOF
• FTICR

21
Steps
Identify Target Proteins for a given stress
condition
DIGE/ICAT/cDNA microarray/predictive data
analysis (Eric Alm)
Identify commercially available antibodies to
similar proteins in E. coli
Antibody Approach
Clone and express genes encoding target proteins
in E. coli with suitable end-tags.
Exogenous Tagged-Protein Approach
Create D. vulgaris mutant strains that express
target proteins with suitable end-tags in-vivo
and use suitable antibiotic markers (Judy Wall)
Endogenous Tagged-Protein Approach
22
Exogenous Tagged-Protein Approach Isolation
A. Target-Protein Generation

• Obtain genomic DNA from wild type D. vulgaris
  cultures
• Design and construct end-primers for protein
  targets as compatible with the GATEWAY system.
• Run and optimize PCR conditions
• Construct appropriate donor vectors and transform
  into appropriate cloning strains
• Isolate plasmid DNA constructs from transformed
  cells
• Verify target gene sequence in constructs
• Pick appropriate destination vectors based on
  desired end-tag
• Create destination constructs and transform into
  appropriate expression strains
• Synthesize tagged-proteins after expression
  optimization
• Purify tagged proteins from host cell proteins
  for hybridization studies

B. Hybridization and Pull-down

• Obtain appropriate cell mass from stress
  condition under study (Terry Hazen)
• Isolate and quantify whole cell lysate
• Quantify purified target protein
• Establish conditions for hybridization Purified
  target protein and whole cell lysate
• Perform pull down of target protein-associated
  proteins using affinity separations on
  appropriate columns
• Run SDS-PAGE gels and stain with appropriate dyes
• Perform in-gel trypsin digestion of observed
  bands

23
Endogenous Tagged-Protein Approach Isolation
A. Target-Protein Generation

• Obtain mutant strains of D. vulgaris expressing
  target proteins with suitable end-tags (Judy
  Wall)
• Replicate media, plating and liquid culture
  growth conditions in Hazen lab
• Isolate single mutant strain colonies and perform
  colony PCRs to verify presence of resistance
  plasmid
• Subculture isolated colonies and re-verify
  presence of plasmid
• Create glycerol stocks of positive subcultures
• Establish growth curves of positive subcultures
  using appropriate media
• Perform appropriate stress experiment on mutant
  strains and obtain cell mass
• Use cell mass from wild type stress condition as
  control

B. Hybridization and Pull-down

• Isolate and quantify whole cell lysate
• Perform pull down of target protein-associated
  proteins using affinity separations on
  appropriate columns
• Run SDS-PAGE gels and stain with appropriate dyes
• Perform in-gel trypsin digestion of observed
  bands

24
Co-Culture Experiment
25
Stage 1
WEEK 1-5
WEEK 3-10
Select Co-culture Organisms
Evaluate Growth in Batch Culture
R4
R2
R1
WEEK 9-12
WEEK 12
yes
Determine Growth Kinetics, Measure Reactants
Products
Biomass Production In Parallel Batch
d1
R6
R7
R3
E2
no
No, try again
No
Close Book
d2
Close Book
Yes
E1
R5
Store Backup samples
Ship to ORNL
For all reports there is an automated email sent
to the teams. Red means a data upload has to
occur. CC does analysis at this point. R1-
Start Stress Book- Record Name, Date, R2- Add
description of condition (protocol) and rationale
to stress book. R3- Add description of
experiment, upload biolog/growth, morphology
data, etc. complete phenomics, add conclusions
from experiment, note person, date, R4- Add
description of why decision was made R5- Upload
key physiology data R6- Upload experimental
design R7- Add comments, upload culture data,
assign sample IDs, delivery point, date shipped
added to ORNL samples. R8- Annotate that
shipment is received. Comments on conditions,
time to analysis, who is doing analysis,
protocols. R9- Upload analysis data. Annotate
with comments and decision.
R8
RNA Analysis
WEEK 13-15
E3
R9
No
Close Book
d3
Yes
Stage 2 or 3
D1- Do we believe we are seeing an interesting
response? D2- (QA/QC verification) D3- Is the RNA
Good? (QA/QC verification)
WEEK 13-19
26
Stage 2
WEEK 19-21
WEEK 22
Select Co-culture
Identify and Test Stressors
R13
R11
R10
WEEK 22-23
WEEK 23
yes
Determine Growth Kinetics, Measure Reactants
Products
Production
d4
R15
R16
R12
E5
no
No, try again
No
Close Book
d5
R14
Close Book
Yes
E4
Store Backup samples
R10- Record Name, Date, R11- Add description of
condition (protocol) and rationale to stress
book. R12- Add description of experiment,
upload biolog/growth, morphology data, etc.
complete phenomics, add conclusions from
experiment, note person, date, R13- Add
description of why decision was made R14- Upload
key physiological data R15- Upload experimental
design R16- Add comments, upload culture data,
assign sample IDs, delivery point, date shipped
added to ORNL samples. R17- Annotate that
shipment is received. Comments on conditions,
time to analysis, who is doing analysis,
protocols. R18- Upload analysis data. Annotate
with comments and decision. R19 - Upload key
physiological data
Ship to ORNL
R17
RNA Analysis
WEEK 23-24
WEEK 25-28
R18
No
Close Book
d6
E6
Yes
D4- Do we believe we are seeing an interesting
response? D5- Did the time series work? (QA/QC
verification) D6- Is the RNA Good? (QA/QC
verification) D7 - Can co-cultures be maintained
in chemostats
Yes
Large Scale Biomass Production
Evaluate Growth In Chemostats /or Biofilm
Reactors
D7
R19
No
WEEK 29-30
Close Book
E7
27
Stage 3
WEEK
WEEK
WEEK
Metabolite Quality Report
Significant Responders Report
Metabolomics
R
R
WEEK
ICAT Proteome
R
R
Ship Samples
Stage 3
Significant Responders Report
R
Proteomic Consistency/ Qual.Report
Diversa Proteome
R
R
Consistency/ Conclusions Report
Sandia DIGE
GO Report
R
R
Phenomics Summary Report
SEM
R
Comparative Stress Report
R
TEM
Yes
EPS Report
Significant Responders Report
R
R
EPS
d
Lipidomics Report
Back to stage 1 or 2
No
R
PLFA
R
Operon/Regulon Report
Microarray Quality Report
Transcriptome
R
R
R- Add shipping date to all other samples. R-
Add receiving dates to samples. Upload Exp.
Design, protocols, contacts, predicted delivery
time.. R Microarray data D Is microarray data
ok? Receive OK from CC before proceeding with
remaining Genomics R Upload data, add comments,
Quality estimates, successful yes or no,
requests for special analysis, etc. Automate
analyses delivered back to stress book.Note If
method fails, there maybe should be a procedure
for requesting a backup sample. Can FGC do
analyses from different exp concomittantly to
increase throughput
GO Report
28
Specific comments for tentative timelines Week
0-1 U of Washington (Stahl lab) Selection and
collation of available data about organisms
tested for syntrophic growth Weeks 1 - 5 U of
Washington (Stahl lab) Demonstrate syntrophic
growth in batch culture Checkpoint (CC) Week
3-10 U of Washington (Stahl lab) Complete
physiological characterization of syntrophic
batch cultures (growth kinetics, biomass yields,
reactant/product profiles). Post data on
biofiles Prepare appropriate monoculture biomass
for controls (substrate-limited chemostat or
batch culture) Week 9-12 U of Washington (Stahl
lab) Prepare batch culture co-culture for ORNL
MA analysis. Transfer co-culture and
mono-cultures to ORNL LBNL(Hazen lab) Conduct
other related assays e.g. IR and PLFA assays Post
data on biofiles ORNL Conduct MA
analysis. Week 13-15 ORNL and U of Washington
(Stahl lab) Obtain MA data for batch co-culture
and mono-culture biomass. Provide data to
computational core group and post on Biofiles
Assess quality of data in general. Email all
and/or post on biofiles. QC of data. Posts
analysis on Biofiles Checkpoint (CC) Modify
conditions and scale of co-culture growth as
required. Repeat microarray analyses as
required. Week 16- 19 U of Washington (Stahl
lab) Prepare large scale biomass for FGC (with
assistance for LBNL?). Distribute Biomass to FGC
and archive additional samples U of Washington
(Stahl lab) Checkpoint (CC). In consultation
with other core groups identify appropriate
co-culture stress conditions.
29
Week 19-21 U of Washington (Stahl lab) /or
LBNL(Hazen lab) Conduct MIC and co-culture curve
assays for batch culture. Checkpoint (CC).
LBNL(Hazen lab) /or U of Washington (Stahl
lab) Prepare mono-culture biomass exposed to
identical stress conditions. ORNL Conduct MA
for their aliquot of the large biomass. Week
22-23 U of Washington (Stahl lab) /or LBNL(Hazen
lab) Provide stressed co-culture biomass to
ORNL for MA analysis Conduct other related assays
e.g. IR and PLFA assays Post data on
biofiles Week 23-24 ORNL and U of Washington
(Stahl lab) Obtain MA data for stressed
co-culture and mono-culture biomass. Provide
data to computational core group and post on
Biofiles Assess quality of data in general.
Email all and / or post on biofiles. QC of data.
Posts analysis on Biofiles Checkpoint (CC)
Modify conditions and scale of co-cultures as
required. Repeat microarray analyses as
required. Week 25-28 U of Washington (Stahl
lab) Evaluate co-culture growth in chemostats
/or biofilm reactor systems Checkpoint (CC). U
of Washington (Stahl lab) Develop biomass at
selected steady-state chemostat (/or biofilm)
growth conditions and transfer to ORNL Week
29-30 ORNL Conduct MA Provide data to
computational core group Comment on any problems
with sample. - Checkpoint (CC) Key decision
point for continued characterization of batch
cultures, chemostat cultures, stress conditions,
determine biomass production needs for other core
groups, selection of additional strains for
characterization of syntrophic growth.
30
Week 31-34 (five weeks) Diversa Conduct
Proteomics 3-4 weeks till final data Post data on
Biofiles LBNL (Keasling lab) conduct ICAT
Proteomics experiments sample prep and run 1-2
weeks data analysis - 2 weeks Post data on
Biofiles Sandia Conduct DIGE experiments 1-3
weeks for sample prep, labeling 2D and obtaining
spots for MALDI 2 weeks for MALDI MS Post data
on Biofiles Parallel efforts during Week 1 4
and 5-10 on underdeveloped Methods Metabolomics
(Keasling lab) Protein-Protein Interactions
(Sandia) Carbohydrate Profiling (Hazen lab
Keasling lab) Week 35 All labs Data
analysis CC Data integration and modeling. All
labs Participate in a Data discussion meeting to
summarize findings What was expected in the
stress? Does our data corroborate these? Trends
observed Up and Down Use of controls to
eliminate growth related changes Co-relating MA
with proteomics data Support from EPS, PLFA, IR
and Metabolite assays Emerging hypothesis
pathways and operons affected by or involved in
stress Implications of the data models for
stress response. Candidates for molecular studies
e.g. Mutations, protein-protein interactions and
Biochemical assays e.g. enzymatic
activity Week 35 Compile above as a
report/manuscript
31
Co-Culture Experimental Rational

• Initial condition experiments and optimization
• Selection of Co-culture organisms
• Selection criteria
• Metabolite and substrate profiles (Availability
  of genome sequences, microarrays, genetic
  systems)
• Known Substate Affinity Characteristics (Ks
  values for key syntrophic substrates (e.g., H2,
  acetate, formate)
• Known Growth Characteristics (Generation times,
  temperature and pH optima,etc.)
• Possible next generation co-cultures
• Alternative methanogenic partner The use of
  methanogen with a lower Ks for hydrogen than
  Methanococcus (e.g., Methanobacterium species)
  should increase free energy available to the
  Desulfovibrio species.
• Deletion mutants
• Deletion of selected uptake or evolution
  hydrogenases
• Deletion of genes for formate production/utilizait
  on
• Deletion of gene involved in EPS synthesis
• Adaptation of co-cultures to syntrophic growth.
• Initial studies suggest that growth of the
  co-culture improves (reduced lag phase, higher
  maximum growth rate) with repeated transfer in
  batch culture. The affect of long term transfer
  on gene expression patterns could provide
  information about the genetic basis for the
  adaptation process and possibly direct continued
  analyses of adapted versus unadapted co-culture
  response to different environmental stresses.
• Identification of appropriate stress conditions
• The co-culture analyses offer the opportunity to
  evaluate direct and indirect stress affects.
  Oxygen and salt stress will have different
  threshold for Methanogen versus desulfovibrio -
  impairment of either partner would result in a
  system-level stress. Addition of hydrogen to
  the culture or reactor headspace would provide
  benefit to the methanogenic partner but reduce
  the free energy available to the Desulfovibrio,
  contributing to stress.