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Team:Purdue/Notebook
From 2012.igem.org
(Difference between revisions)
| Line 343: | Line 343: | ||
<h1> Characterization and Experimental Design </h1> | <h1> Characterization and Experimental Design </h1> | ||
| - | <h5> | + | <h5> Meeting 7/2/12 |
| + | |||
| + | List of What Needs to get Done this Week: | ||
| + | <ul> | ||
| + | <li> Research protocols on setting up and running experiments using biofilms </li> | ||
| + | <li> Research flow and static protocols for biofilms </li> | ||
| + | <li> Research ways to characterize silica matrix and acquire quantifiable data </li> | ||
| + | <li> Create timelines for each assay researched </li> | ||
| + | <li> Order reagents and get training on any equipment needed for assays </li> | ||
<h1> Modeling </h1> | <h1> Modeling </h1> | ||
Revision as of 20:49, 2 July 2012
You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.
General Meeting Notes
Monday, May 15
List of useful contacts on the google doc, including DowAgro
Begin to list the different devices/constructs that will be used in our project
Attachment (adhesion)
Filtration
- Modularize the sequence so we can test individually (e.g. but GFP, RFP, YFP at the end of each segment – construct silica binding protein first with constitutive promoter/repressible promoter to produce promoter and make sure it does what you think it should)
- Investigate multiple silica binding protein (surface protein – silica binding peptide);must choose several top candidates for each element.
Hierarchy
Perfecting the FFL
- And (low affinity, not dimer), Or (high affinity)
- Schematic Diagram
Modeling and Experimental
- Communication in terms of data (e.g. kinetic parameters)
- Review Characterization Data Sheets (look in the DropBox for an uploaded link from Sean )
- Strong integration of modeling translates to a strong performance in the competition.
List of things we need
- Competent Cells (Laris Avramova (core molecular biologist, 222), Tarun (electron microscopy)may have the needed cells)
- Antibiotics (AMP, tetracycline)
- Enzymes (Pst1, Xba1, EcoR1, Spe1, Ligase, polymerase/PCR reagents, T5exonuclease )
- Parts (available in the registry)
- Constitutive promoter (orthogonal t7 promoter)
- Signaling Promoters (investigate the precedent for construction FFL)
- RBS –(B0034)
- Thermodynamic models for designing RBSs, etc (Voights model)
- Terminators
- Proteins Transcription Factors
Monday, May 21
We're all looking forward to an exciting iGEM summer! Our SURF students have just arrived and are gradually being introduced to synthetic biology and iGEM.
Sean gave a crash course on synthetic biology to Mrudula, Rachel, Amanda and August. The powerpoint is available here, compiled by our wonderful graduate mentor, Janie.
Tuesday, May 22
First Journal Club Meeting - Identified the reducible elements of our system
Detailed outline of the project to the SURF students
What is the advantage of using this entire process? Is not it kind of roundabout?
For the NEXT MEETING Tuesday 29th May :
Identify, in these teams:
- a. What Adhesion system we want to use (Amanda, Peter, Mrudula)
- b. Which silica-binding system we want to use (Rachel, August)
- c. Control Elements (Max, Mrudula, Rachel, Sean)
- d. Find strain auxotrophic for RSC (gene which breaks down arabinose) (Jim)
Announcements:
- Be ready to explain your assigned element of the project (starting generally and moving more specifically)
- Read the introductory/background elements of the thesis that Dr. Rickus will put in the dropbox
General Announcements
- Be ready to work the BioBuilder HighSchool Teacher iGEM workshop during the week of June 4th – June 8th (more details to come)
- Everyone is welcome to visit Drs. Rickus and Clase’s lab group meeting on Thursday at 12PM
Thursday May 24th
Update on the human practice component available through the Powerpoint
Tuesday, May 29
Overview of small group presentations
Adhesion Proteins – Amanda, Mrudula, and Peter
- Ag43 : [University of Queensland 2009] PROS: makes chains instead of aggregates, works well in flow, in the registry, abiotic adherence CONS: not concomitant
- TibA: PROS: modular, concomitant, auto-transport CONS: not in the registry
- AIDA-1: PROS: binds Ag43 and self, in registry, higher shear tolerance CONS: only expressed in certain cells, blocked by Fimbriae (due to length)
- FimA-H: [Michigan 2010] PROS: forms chains, compatible w/ E. coli, shear resistant, grows in constant flow, binds well to glycoproteins CONS: inhibits function of other proteins
- Curli: [Lyons 2011] PROS: well characterized in registry, amyloid fibers CONS: inhibits Ag43 and AIDA-1
Decision:
- primary: AIDA-1 [order from registry and improve bad sequencing or re-synthesize]
- secondary (if AIDA-1 proves unfeasible): TibA [with goal of characterizing part, must synthesize]
Silica Binding Proteins – Rachel and August
- INP – Silicatein [MN 2011] – PROS: CONS: very large, no data on viability, not biobrick compatible.
- OmpA-Silicatein alpha fusion – PROS: shorter than INP-Silicatein, active in neutral pH, no illegal sites, known to work CONS: must construct fusion peptide vector NOTES: optimum at low temperatures [OmpA - K103006]
- R5 peptide: PROS: active at neutral pH, small, has been used in E. coli CONS: part of a larger protein (no silaffin post-translational modifications), contains EcoRI site
Modeling the Network Motif - Max, Mrudula, Rachel, and Sean
- Modeled the simplified system
- Matlab and MathCad Model
- Need concrete entry parameters for more robust models
For Next week:
- Construct your parts in DNA 2.0 and anything in registry start detailing
Wednesday, May 30
In Lab:
- Cleaned and organized the laboratory space
- Ordered laboratory suppleies
- Made LB agar plates and LB liquid media with ampicillin
- Created the adhesions and SBP devices in silico using Gene Designer by DNA 2.0
Thursday, May 31
Attended Rickus and Porterfield laboratory group meeting
- Each person introduced themselves and their research
- Decided on the use of future meetings
Researched primer design and began to design primers. These primers will be used for PCR to perform the Gibson assembly method
Friday, June 1
Performed Cell Transformations of the following parts:
- Lac promotor and CFP generator (BBa_I13601)
- PoPs receiver (BBa_F2620)
- Tet repressor (BBa_C0040)
- Tet promotor with green fluorescent protein (BBa_I13522)
The transformation protocol can be found here
Completed Primer Sequences for surface expression protein
Monday, June 4
Welcomed Bio Builders workshop participants
Janie gave a powerpoint on the basics of synthetic biology to the workshop participants
Amanda, August, Max, Mrudula, and Rachel helped the participants with an experiment based on MIT's 2006 iGEM project, a banana scent generator
Mrudula and Amanda completed minipreps for the previously transformed parts. The results of the minipreps are shown below.
- insert results
- insert results
- insert results
- insert results
Tuesday, June 5
Amanda, August, Mrudula, Rachel, and Soo spent the morning watching presentations on abstraction and devices with the Bio Builder workshop participants
Soo gave a presentation to the participants on how to use TinkerCell
The iGEM team helped the Bio Builder participants to transform green and purple fluorescent protein into E. coli
Created a timeline for the rest of the summer as seen below
- Week 3:
- Design and order primers for Gibson assembly
- Order reagents
- Complete a miniprep for the previously transformed parts
- Select plasmid backbones
- Week 4:
- PCR parts for Gibson assembly
- Complete Gibson assembly
- Decide on which parts will be synthetic
- What devices can be synthesized or should we synthesize individual parts?
- Create competent cells
- Week 5:
- Complete plasmid midi and mega preps
- Transform parts into expression vectors
- Week 6:
- Complete functional analysis of protein expression
- Construct the full system
- Week 7:
- Build the flow system for biofilm establishment
- Complete functional analysis of biofilm thickness
- Week 8-10:
- Iterate and improve on flow setup
Wednesday, June 6
Sean gave a presentation to the Bio Builder participants on 3A and Gibson assembly
Sean lead a 3A assembly lab with the participants
Worked on designing primers
Thursday, June 7
Continued to design primers
Monday, June 11
We attempted to optimize our primers using online guidlines and software.
- Here is a website that details primer design.
- We used a calculator and an Oligo Analyzer which can calculate the liklihood of occurances such as hairpins and dimers.
Upon analysis, we found that the primers that would be required to use Gibson assembly were not ideal. In some cases, the forward and the reverse primers did not have Tm values that were close enough to each other. We also had difficulty with making our primers a reasonable length. Furthermore, many of the primers had a high probability (Gibson free energy value lower than zero) of froming dimers or hairpins. Finally, we deicided that it would most likely be more successful to use 3A assembly instead of Gibson.
In order to save time and ensure the accuracy of the sequence, we decided to synthesize instead of assemble the whole silica making device in a high copy plasmid.
Welcomed Rubeena to the iGEM team. We are looking forward to working with her!
Tuesday, June 12
insert meeting minutes
Wednesday, June 13
August, Mrudula, and Rachel were trained on how to use the flow cytometer
Made competent cells (DH5α). The protocol for making competent cells is found here
Thursday, June 14
Rachel had a demonstration on how to make silica. The procedure is shown here.
We welcomed Chris to the iGEM team. We know he will be a great asset to the team!
We had an overview meeting for Rubeena and Chris to help them catch up on the project.
Friday, June 15
We transformed all of the parts needed for the adhesion device. These included the following
- AIDA-1 (BBa_K257018)
- Plasmid (pSB1AK3)
- pTet (BBa_R0040)
- RBS (BBa_B0034)
- Terminator (BBa_B0015)
- Plasmid (pSB1AC3)
- CFP (BBa_E0020)
insert meeting minutes
Saturday, June 16
Removed the following parts from the incubator and placed in the refrigerator with parafilm wax
- BBa_E00032 (GFP device from plates)
- Bba_
Sunday, June 17
We reevaluated our previous decision to synthesize the silica making device. We have now decided to use Gibson assembly to create the OmpA-silicatein fusion sequence and 3A assembly for the rest of the device. We believe that this will save money and, considering the time taken to order and deliver the sequence, will not take any more time than synthesizing.
Monday, June 18
Completed minipreps on recently transformed parts. The results are shown below.
- GFP: 17.5ng/μL and 7.6 ng/μL
- AIDA-1: 8.7ng/μL and 6ng/μL
- pSB1AC3: 5.2ng/μL snd 4.2ng/μL
- CFP: 13.6ng/μL and 12.7ng/μL
- RBS (BBa_B0032): 24.9ng/μL and 24.2ng/μL
- PoPs: 107.8ng/μL and 131.4ng/μL
- pSB1AK3: 7.0ng/μL
- Terminator: 11.5ng/μL
- RBS (BBa_B0034): 21.0ng/μL and 21.5ng/μL
- pTet: 5.2ng/μL
These concentrations are too low to proceed with 3A assembly. Therefore, we are going to grow more of the transformed bacteria, and we are going to perform more minipreps.
Tuesday, June 19
August completed a second round of minipreps. The results are listed below.
- RBS (BBa_B0034): 14.8ng/μL
- RBS (BBa_C0040): 5.0ng/μL
- CFP: 9.0ng/μL
- PoPs: 14.9ng/μL
- AIDA-1: 7.0ng/μL
Wednesday, June 20
Due to the low yields from our previous tranformations and minipreps, we have decided to attempt our transformations with a new protocol. The new protocol can be found here
Thursday, June 21
insert meeting notes
Friday, June 22
insert meeting notes
Saturday, June 23
insert meeting notes
Sunday, June 24
insert meeting notes
Monday, June 25
insert meeting notes
Tuesday, June 26
insert meeting notes
Wednesday, June 27
insert meeting notes
Thursday, June 28
insert meeting notes
Friday, June 29
insert meeting notes
Saturday, June 30
insert meeting notes
Sunday, July 1
insert meeting notes
Monday, July 2
insert meeting notes
At this point in, note for each committee will be housed in their committee subsections (see below)
Characterization and Experimental Design
Meeting 7/2/12
List of What Needs to get Done this Week:
- Research protocols on setting up and running experiments using biofilms
- Research flow and static protocols for biofilms
- Research ways to characterize silica matrix and acquire quantifiable data
- Create timelines for each assay researched
- Order reagents and get training on any equipment needed for assays
Modeling
Modeling Biofilms
Levels of Investigation
- How biofilm responds to shear, flow, temp, surface attachment, etc
- How Silica traps the particle during flow
- How Curli adheres and how OmpA-Silicatein Alpha polymerizes Silica/How the silica crystalizes (introduction of Salicylic Acid)
- How protien expression responds to external and metabolic variation (e.g. introduction of IPTG to system)
- How constructs work with each other/controls systems
- How RBS/ Promoter effects protein expression
Distribution of Modeling
Matlab
- Protein Production
- Feed Forward Loop Control Structure
TinkerCell
- Quorum sensing
- Feed Forward Loop
JMP
- Experimental Design and Characterization Experiments
Comsol/Other
- Waterflow and shear force on final system
- Silica formation
References and Citations
insert meeting notes
Wetlab Work
insert meeting notes
Wiki
insert meeting notes
Filtration
- Modularize the sequence so we can test individually (e.g. but GFP, RFP, YFP at the end of each segment – construct silica binding protein first with constitutive promoter/repressible promoter to produce promoter and make sure it does what you think it should)
- Investigate multiple silica binding protein (surface protein – silica binding peptide);must choose several top candidates for each element.
Hierarchy
Perfecting the FFL
- And (low affinity, not dimer), Or (high affinity)
- Schematic Diagram
Modeling and Experimental
- Communication in terms of data (e.g. kinetic parameters)
- Review Characterization Data Sheets (look in the DropBox for an uploaded link from Sean )
- Strong integration of modeling translates to a strong performance in the competition.
List of things we need
- Competent Cells (Laris Avramova (core molecular biologist, 222), Tarun (electron microscopy)may have the needed cells)
- Antibiotics (AMP, tetracycline)
- Enzymes (Pst1, Xba1, EcoR1, Spe1, Ligase, polymerase/PCR reagents, T5exonuclease )
- Parts (available in the registry)
- Constitutive promoter (orthogonal t7 promoter)
- Signaling Promoters (investigate the precedent for construction FFL)
- RBS –(B0034)
- Thermodynamic models for designing RBSs, etc (Voights model)
- Terminators
- Proteins Transcription Factors
Monday, May 21
We're all looking forward to an exciting iGEM summer! Our SURF students have just arrived and are gradually being introduced to synthetic biology and iGEM.
Sean gave a crash course on synthetic biology to Mrudula, Rachel, Amanda and August. The powerpoint is available here, compiled by our wonderful graduate mentor, Janie.
Tuesday, May 22
First Journal Club Meeting - Identified the reducible elements of our system Detailed outline of the project to the SURF students What is the advantage of using this entire process? Is not it kind of roundabout?
For the NEXT MEETING Tuesday 29th May :
- a. What Adhesion system we want to use (Amanda, Peter, Mrudula)
- b. Which silica-binding system we want to use (Rachel, August)
- c. Control Elements (Max, Mrudula, Rachel, Sean)
- d. Find strain auxotrophic for RSC (gene which breaks down arabinose) (Jim)
Identify, in these teams:
Announcements:
- Be ready to explain your assigned element of the project (starting generally and moving more specifically)
- Read the introductory/background elements of the thesis that Dr. Rickus will put in the dropbox
General Announcements
- Be ready to work the BioBuilder HighSchool Teacher iGEM workshop during the week of June 4th – June 8th (more details to come)
- Everyone is welcome to visit Drs. Rickus and Clase’s lab group meeting on Thursday at 12PM
Thursday May 24th
Update on the human practice component available through the Powerpoint
Tuesday, May 29
Overview of small group presentations
Adhesion Proteins – Amanda, Mrudula, and Peter
- Ag43 : [University of Queensland 2009] PROS: makes chains instead of aggregates, works well in flow, in the registry, abiotic adherence CONS: not concomitant
- TibA: PROS: modular, concomitant, auto-transport CONS: not in the registry
- AIDA-1: PROS: binds Ag43 and self, in registry, higher shear tolerance CONS: only expressed in certain cells, blocked by Fimbriae (due to length)
- FimA-H: [Michigan 2010] PROS: forms chains, compatible w/ E. coli, shear resistant, grows in constant flow, binds well to glycoproteins CONS: inhibits function of other proteins
- Curli: [Lyons 2011] PROS: well characterized in registry, amyloid fibers CONS: inhibits Ag43 and AIDA-1
Decision:
- primary: AIDA-1 [order from registry and improve bad sequencing or re-synthesize]
- secondary (if AIDA-1 proves unfeasible): TibA [with goal of characterizing part, must synthesize]
Silica Binding Proteins – Rachel and August
- INP – Silicatein [MN 2011] – PROS: CONS: very large, no data on viability, not biobrick compatible.
- OmpA-Silicatein alpha fusion – PROS: shorter than INP-Silicatein, active in neutral pH, no illegal sites, known to work CONS: must construct fusion peptide vector NOTES: optimum at low temperatures [OmpA - K103006]
- R5 peptide: PROS: active at neutral pH, small, has been used in E. coli CONS: part of a larger protein (no silaffin post-translational modifications), contains EcoRI site
Modeling the Network Motif - Max, Mrudula, Rachel, and Sean
- Modeled the simplified system
- Matlab and MathCad Model
- Need concrete entry parameters for more robust models
For Next week:
- Construct your parts in DNA 2.0 and anything in registry start detailing
Wednesday, May 30
In Lab:
- Cleaned and organized the laboratory space
- Ordered laboratory suppleies
- Made LB agar plates and LB liquid media with ampicillin
- Created the adhesions and SBP devices in silico using Gene Designer by DNA 2.0
Thursday, May 31
Attended Rickus and Porterfield laboratory group meeting
- Each person introduced themselves and their research
- Decided on the use of future meetings
Researched primer design and began to design primers. These primers will be used for PCR to perform the Gibson assembly method
Friday, June 1
Performed Cell Transformations of the following parts:
- Lac promotor and CFP generator (BBa_I13601)
- PoPs receiver (BBa_F2620)
- Tet repressor (BBa_C0040)
- Tet promotor with green fluorescent protein (BBa_I13522)
The transformation protocol can be found here
Completed Primer Sequences for surface expression protein
Monday, June 4
Welcomed Bio Builders workshop participants
Janie gave a powerpoint on the basics of synthetic biology to the workshop participants
Amanda, August, Max, Mrudula, and Rachel helped the participants with an experiment based on MIT's 2006 iGEM project, a banana scent generator
Mrudula and Amanda completed minipreps for the previously transformed parts. The results of the minipreps are shown below.
- insert results
- insert results
- insert results
- insert results
Tuesday, June 5
Amanda, August, Mrudula, Rachel, and Soo spent the morning watching presentations on abstraction and devices with the Bio Builder workshop participants Soo gave a presentation to the participants on how to use TinkerCell The iGEM team helped the Bio Builder participants to transform green and purple fluorescent protein into E. coli
- Week 3:
- Design and order primers for Gibson assembly
- Order reagents
- Complete a miniprep for the previously transformed parts
- Select plasmid backbones
- Week 4:
- PCR parts for Gibson assembly
- Complete Gibson assembly
- Decide on which parts will be synthetic
- What devices can be synthesized or should we synthesize individual parts?
- Create competent cells
- Week 5:
- Complete plasmid midi and mega preps
- Transform parts into expression vectors
- Week 6:
- Complete functional analysis of protein expression
- Construct the full system
- Week 7:
- Build the flow system for biofilm establishment
- Complete functional analysis of biofilm thickness
- Week 8-10:
- Iterate and improve on flow setup
- Here is a website that details primer design.
- We used a calculator and an Oligo Analyzer which can calculate the liklihood of occurances such as hairpins and dimers.
- AIDA-1 (BBa_K257018)
- Plasmid (pSB1AK3)
- pTet (BBa_R0040)
- RBS (BBa_B0034)
- Terminator (BBa_B0015)
- Plasmid (pSB1AC3)
- CFP (BBa_E0020)
- BBa_E00032 (GFP device from plates)
- Bba_
- GFP: 17.5ng/μL and 7.6 ng/μL
- AIDA-1: 8.7ng/μL and 6ng/μL
- pSB1AC3: 5.2ng/μL snd 4.2ng/μL
- CFP: 13.6ng/μL and 12.7ng/μL
- RBS (BBa_B0032): 24.9ng/μL and 24.2ng/μL
- PoPs: 107.8ng/μL and 131.4ng/μL
- pSB1AK3: 7.0ng/μL
- Terminator: 11.5ng/μL
- RBS (BBa_B0034): 21.0ng/μL and 21.5ng/μL
- pTet: 5.2ng/μL
- RBS (BBa_B0034): 14.8ng/μL
- RBS (BBa_C0040): 5.0ng/μL
- CFP: 9.0ng/μL
- PoPs: 14.9ng/μL
- AIDA-1: 7.0ng/μL
- Research protocols on setting up and running experiments using biofilms
- Research flow and static protocols for biofilms
- Research ways to characterize silica matrix and acquire quantifiable data
- Create timelines for each assay researched
- Order reagents and get training on any equipment needed for assays
- How biofilm responds to shear, flow, temp, surface attachment, etc
- How Silica traps the particle during flow
- How Curli adheres and how OmpA-Silicatein Alpha polymerizes Silica/How the silica crystalizes (introduction of Salicylic Acid)
- How protien expression responds to external and metabolic variation (e.g. introduction of IPTG to system)
- How constructs work with each other/controls systems
- How RBS/ Promoter effects protein expression
- Protein Production
- Feed Forward Loop Control Structure
- Quorum sensing
- Feed Forward Loop
- Experimental Design and Characterization Experiments
- Waterflow and shear force on final system
- Silica formation
