# Presentation: 504952

## Making a Stress Book

**Making a Stress Book**

**Scientific Flow Charts for Genomics:GTL Project at**
**Lawrence Berkeley National Laboratory**

## no

- Perturbation

- Growth and
- MIC

- d1

- R2

**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.

- R1

- E1

- R3

- E2

- 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)

- Close Book

- Design
- Time/Dose
- Series (S-FTIR)

- R4

- R6

- yes

- Execute
- Time Series

- R7

- Store
- Backup
- samples

- d2

- R8

- R9

- No, try again

- Close Book

- No

- Yes

- Ship to
- ORNL

- RNA Analysis

- d3

- Stage 2

- Yes

- E3

- Close Book

- No

***WEEK 1-2***

***WEEK 3-4***

***WEEK 5-6***

***WEEK 7***

***WEEK 7-8***

***WEEK 9***

- R5

- 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.

## Stage 2 

- SEM

- TEM

- PLFA

- Metabolomics

- R19

- Stage 2

- R9

- **Separate and
- Ship
- Samples

**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**

- R15

- ICAT Proteome

- R20

- Diversa
- Proteome

- Sandia
- DIGE

- Transcriptome

- R10

- R11

- R12

- R13

- R14

- R21

- R22

- R23

- R18

- Proteomic
- Consistency/
- Qual.Report

- Microarray
- Quality
- Report

- Operon/Regulon
- Report

- Significant
- Responders
- Report

- GO Report

- Significant
- Responders
- Report

- GO Report

- Phenomics
- Summary
- Report

- Metabolite
- Quality
- Report

- Significant
- Responders
- Report

- Consistency/
- Conclusions
- Report

- Comparative
- Stress
- Report

***WEEK 10-20***

***WEEK 8-10***

***WEEK 21-22***

***WEEK 23***

- d4

- Yes

- No

- Back to stage 1

- EPS

- R16

- R17

- R24

- R25

- EPS
- Report

- Lipidomics
- Report

## 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)

## 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

## Stressors

| Oxygen | The most difficult but most important.  | 1 |
| --- | --- | --- |
| Nitrite/Nitrate | Perhaps both | 1 |
| Osmotic/Ionic | Classical stressors | 1 |
| pH: High and Low | Classical but difficult stressors | 1 |
| Chromium VI | Only at 5 DOE Sites | 2 |
| Cadmium (other heavy metals | Precipitates make things difficult | 2 |
| Phosphate starvation | Classical stressor | 1 |
| Sulfate Starvation | Stress for our bugs | 1 |
| Carbon Starvation | Classical Stressor | 1 |
| Uranyl (6) | Sample handling issues | 2 |
| Heat Shock | Classical Stressor | 1 |

- 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.

## Environmental Characterization and Sequencing

## no

- Site

- Site
- Data

- d1

- R2

**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 EC&S 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.]]

- R1

- E1

- R3

- E2

- D1- Is this site interesting and relevant to stressors we are studying?
- D2- Are the samples of good quality?  (QA/QC verification)

- Close Book

- Design
- Field Sampling
- Plan

- R4

- R6

- yes

- Execute
- Field Sampling

- R7

- Store
- Backup
- samples

- d2

- No, try again

- Close Book

- No

- Yes

- Stage 2

***WEEK 1-3***

***WEEK 4-5***

***WEEK 5-6***

***WEEK 7***

***WEEK 8***

- R5

- 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.

- Ship to
- LBNL

## Stage 2 

- Physical

- Chemical

- PLFA

- UW/MU
- Enrichments

- R16

- Stage 2

- R8

- Ship
- Samples

**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.**

- R13

- MU/ORNL
- F Probe/MA

- R17

- Diversa
- Clone Libraries

- LBNL/JGI
- 16s Microarray

- R9

- R10

- R11

- R12

- R18

- R19

- R20

- Ecogenomics
- Report

- Significant
- Stressors
- Report

- Functional
- Population
- Report

- Functional
- Activity
- Report

- Geochemical
- Report

- Stimulated
- Activity
- Report

- Community
- Structure
- Report

- Conceptual
- Biogeochemical
- Model
- Report

- Comparative
- Stress
- Report

***WEEK 10-14***

***WEEK 8-9***

***WEEK 14-15***

***WEEK 16***

- Direct Counts

- R14

- R15

- R22

- R23

- Biomass
- Report

- Lipidomics
- Report

## 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

## 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
- 	Compile above as a report/manuscript

## Deletion Project

## No

- Choose
- Marker

- Obtain
- Sequence

- Design
- Primers

- Order
- Primers

- PCR1

- D1

- Repeat PCR1

- Yes

- D2

- Store

- Yes

- Barcode

- Common

- Gel Purify

- Choose Gene
- for Deletion

- Obtain Up/Down-
- Stream Sequence

- Design
- Primers

- Order
- Primers

- PCR2 and PCR3

- D3

- PCR4

- Yes

- Repeat PCR2
- and PCR3

- Yes

- D4

- Gel Purify

- Ligate into
- Cloning Vector

- Ligate into
- Mut. Vector

- Yes

- Repeat PCR4

- Change PCR
- Conditions

- Yes

**Close**
**Book**

- Yes

- R 1.  Construct mutagenic plasmid
- R 2.  Construct mutant

- 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.

- R 2

- 1 Day *

- 7-14 Days

- 3 - 5 Days

- B

- B

- B

- R 1

## PCR4

- 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

- ATG

- TAA

**Ab****1****R**

**PCR4**

- Common A

- Common B

- Barcode 1

- Barcode 2

## No

- Electrotransformation
- into SRB

- Conjugation into SRB

- Purify Ab1R CFU

- Purify Ab1R CFU

- R 2

- D5

- Yes

- Yes

- Yes

- Confirm Plasmid
- Mutation

- R 2.  Construct mutant: First Recombination for Plasmid Integration
- R 3.  Construct mutant: Second Recombination

- PCR Junction Product

- Southern of
- Digested Chromosome

- D6

- R 3

- R 2

- B

**Close**
**Book**

- 5-7 Days*

- 7 Days

- 1-3 Days

## Yes

- R 3

- R 3.  Construct mutant: Second Recombination
- R4.  Characterize Mutant

- Enrich SucroseR

- Plate SucroseR
- and Ab1R

- Screen Plasmid Ab2S

- No

- Confirm Plasmid
- Mutation

- PCR Junction Product

- Southern of
- Digested Chromosome

- D7

- R 4

- R 3

- B

- Grow and
- Make Glycerol Stock

- Growth Curves:
- Carbon sources
- Stress responses

- Ship to LBL for Biomics

- B

- 1-3 Days

- 7 Days *

- 3 Days

- 7-30 Days

**Close**
**Book**

- No

## Gene deletion in D.vulgaris:  Stages in creating knockout mutants 

- Select genes

- PCR

- Obtain TAGs
-    from CC

- Design and order primers

- PCR

- Clone in suicide vector

- Design and develop suicide vector

- Set up transformation protocols
- Conjugation
- Electroporation

- Antibiotics

- Methods development

- Gene deletion experiment

- sequence

- B

- B

- B

- B

- B

- =  post on Biofiles

- 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

- Start point

- 1-2 weeks

- 2-3 weeks

- > 1 week

- B

## Transform E.coli donor - S17-1

- Transform *E.coli* donor - S17-1

- Grow Dv wt

- Grow S17-1

- Prepare LS4D plates and media

- Set up conjugation

- Screen for colonies

- grow

- colony pcr

- southern

- biolog

- Stocks

- B

- B

- B

- B

- 1 week

- 1 week

- 1 - 2 weeks

- 1- 2 weeks

- 2 - 4 weeks

## Molecular Complexes

## Molecular Complexes:

** ****Isolation: Three Parallel Approaches**
**Antibody pull down approach**
**Tagged Protein approach**
**Endogenous**
**Exogenous**

** ****Identification: **
**SDS-PAGE MALDI-MS**
**Nano-LC QTOF**
**FTICR**

## 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.

- Create *D. vulgaris* mutant strains that express target proteins with suitable end-tags
*in-vivo *and use suitable antibiotic markers (*Judy Wall)*

**Exogenous Tagged-Protein  **
**Approach**

**Endogenous Tagged-Protein  **
**Approach**

## Exogenous Tagged-Protein  Approach: Isolation

- 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

**A. Target-Protein Generation**

**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

## 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

## Co-Culture Experiment

## no

- Organisms

- Evaluate
- Growth in Batch Culture

- d1

- R2

**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.

- R1

- E1

- R3

- E2

- D1- Do we believe we are seeing an interesting response?
- D2- (QA/QC verification)
- D3- Is the RNA Good? (QA/QC verification)

- Close Book

- Determine Growth
- Kinetics, Measure
- Reactants &
- Products

- R4

- R6

- yes

- Biomass Production
- In Parallel  Batch

- R7

- Store
- Backup
- samples

- d2

- R8

- R9

- No, try again

- Close Book

- No

- Yes

- Ship to
- ORNL

- RNA Analysis

- d3

- Stage 2 or 3

- Yes

- E3

- Close Book

- No

***WEEK 1-5***

***WEEK 3-10***

***WEEK 9-12***

***WEEK 12***

***WEEK 13-15***

***WEEK 13-19***

- R5

- Stage 1

## no

- Identify and Test
- Stressors

- d4

- R11

- 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

- R10

- E4

- R12

- E5

- 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

- Close Book

- Determine Growth
- Kinetics, Measure
- Reactants &
- Products

- R13

- R15

- yes

- Production

- R16

- Store
- Backup
- samples

- d5

- R17

- R18

- No, try again

- Close Book

- No

- Yes

- Ship to
- ORNL

- RNA Analysis

- d6

- Yes

- E6

- Close Book

- No

***WEEK 19-21 ***

***WEEK 22***

***WEEK 22-23***

***WEEK 23***

***WEEK 23-24 ***

***WEEK 29-30***

- R14

- Stage 2

- Evaluate Growth
- In Chemostats
- &/or Biofilm Reactors

- Large Scale Biomass
- Production

- E7

- Close Book

- R19

**No**

***WEEK 25-28 ***

- D7

**Yes**

## Stage 3 

- SEM

- TEM

- PLFA

- Metabolomics

- R

- Stage 3

- R

- Ship
- Samples

- 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**

- R

- ICAT Proteome

- R

- Diversa
- Proteome

- Sandia
- DIGE

- Transcriptome

- R

- R

- R

- R

- R

- R

- R

- R

- R

- Proteomic
- Consistency/
- Qual.Report

- Microarray
- Quality
- Report

- Operon/Regulon
- Report

- Significant
- Responders
- Report

- GO Report

- Significant
- Responders
- Report

- GO Report

- Phenomics
- Summary
- Report

- Metabolite
- Quality
- Report

- Significant
- Responders
- Report

- Consistency/
- Conclusions
- Report

- Comparative
- Stress
- Report

***WEEK***

***WEEK***

***WEEK***

***WEEK***

- d

- Yes

- No

- Back to stage 1 or 2

- EPS

- R

- R

- R

- R

- EPS
- Report

- Lipidomics
- Report

- Stage 3

## 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.

## 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.

## 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

## 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/utilizaiton
- 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.