Team:Purdue/Notebook
From 2012.igem.org
![](https://static.igem.org/mediawiki/2012/3/3d/Purdue_fountain.jpg)
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. put a fluorescent protein coding sequence at the end of each segment – construct silica binding protein first with constitutive promoter/repressible promoter to produce fluoresent protein and make sure it does what you think it should)
- Investigate multiple silica binding protein (SBP);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
-
- 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
Today, we transformed pLac, TetR, terminator (BBa_B0015), RBS (BBa_B0034), pSB1A3, and pSB1C3.
The minipreps gave the following results
- RBS: 14.8ng/μL
- TetR: 5.0ng/μL
- CFP: 9.0ng/μL
- PoPs: 14.5ng/μL
- AIDA-1: 7.0ng/μL
Thursday, June 21
Due to the low yield of our transformations and minipreps, we have now decided to troubleshoot our transformation procedures. To do this, we will be doing a transformation with Tarun, a graduate mentor to the team. We will be using Tarun's procedures and supplies. We will also have a control plate that will contain cells that had no DNA inserted. If these transformations succeed, then we will have narrowed the possibilities of what went wrong with our procedures. Tarun's transformation procedure can be found on the protocol page. We performed these test transformations with BBa_I13602 (CFP).
Upon closer inspection of the AIDA-1 biobrick (BBa_K257018), we have found the following problems
- the sequencing is bad
- there are large gaps from the sequencing as compared to what was documented. Specifically, the sequenced biobrick was missing approximately 500bp
- the sequence was supposed to be 1326bp long. We have decided that it would cost too much to have the biobrick sequenced ourselves
- the gel is bad
For the above reasons, we have decided to not use AIDA-1. After reevaluating the other adhesion options, we have decided to use Curli (BBa_342003). Due to its inconsistant sequencing, we have decided to have the part found in the kit sequenced ourselves.
Friday, June 22
Due to our increasing size, we have decided to divide into committees. These committees will help us divide the workload fairly and ensure that all tasks are being completed in a timely manner. The committees and their members are listed below. An asteric indicates the committee head.
- Wetlab
- OmpA-silicatein
*August
Haefa
- Curli
*Rubeena
- Purchasing
*Peter
- Human Practices
*Peter
*Max
- Experimental Design/Characterization
*Amanda
*Mrudula
Chris
Arthi
Rachel
Jim
- Project Planning
*Namita
- Wiki
*Rachel
Sean (background)
We checked our test transformations from yesterday. None of the plates showed growth.
Monday, June 25
The foci of this week are to troubleshoot our transformations and research ways to characterize our constructs once they are assembled. August and Mrudula will be in lab troubleshooting. Amanda and Mrudula will be resarching ways to characterize a biofilm. Rachel will be researching ways to characterize a silica coat.
Friday, June 29
We went to Dow Agro, one of our sponsors, to give them an overview of our project. The powerpoint presentation we gave can be found Powerpoint. After we gave our presentation, we asked the Dow representative, Ryan, if he had any guidance with concern to Gibson assembly.
Saturday, June 30
Part of the team volunteered at the high school Jamboree. We saw many amazing teams with remarkable ideas! Congratulations to all the teams for their hard work over the past semester, and good luck as future iGEMers!
Sunday, July 1
We attempted another test transformation, this time using bought Excel 10β competent cells. We had both a negative and positive control. We also plated different concentrations of the final solution to see if we were over or under plating. We also used agar plates with ampicillin that Tarun made. We used store bought SOC. The part used was BBa_J04450.
Monday, July 2
The tranformations performed yesterday were successful. Both the positive control and the plaes with our part showed growth. The transformation efficiency was extremely high. We got single colonies on the plates with 50μL and overgrowth on the plates that had 150μL. We will only plate at 50μL from now on.
Due to time constraints, we have decided to synthesize both of our constructs. In parallel to characterization of the synthesized constructs, we are going to assemble both constructs using Gibson assembly. The assembles constructs will differ from the synthesized constructs in that the promoter will be a PoPs device and there will be a fluorescent protein on each. In this way, we can find if the addition of these two characterization parts will affect the functionality of our constructs.
The synthesized constructs were ordered from DNA 2.0. We are having two versions of the Curli construct made--one with a fluorescent reporter and one without. Pictures of the Gene Designer (DNA 2.0 software) renditions of the three constructs are given below.
We transformed Curli so that we can have it sequenced soon
Tuesday, July 3
We are now awaiting the delivery of our synthesized constructs. In preparation, we are researching the varying types of assays that we may be able to use. You can find information about the different assays we researched can be found here.
Silicatein-Alpha Characterization
We have decided to use scanning electron microscopy (SEM)< x-ray diffraction (XRD), and a live dead assay (specifically, the Baclight Bacterial Viability Kit from Invitrogen).
Further assays will be done once the two constructs are used within the same bacteria.
Filtration
- Modularize the sequence so we can test individually (e.g. put a fluorescent protein coding sequence at the end of each segment – construct silica binding protein first with constitutive promoter/repressible promoter to produce fluoresent protein and make sure it does what you think it should)
- Investigate multiple silica binding protein (SBP);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
- 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
- RBS: 14.8ng/μL
- TetR: 5.0ng/μL
- CFP: 9.0ng/μL
- PoPs: 14.5ng/μL
- AIDA-1: 7.0ng/μL
- the sequencing is bad
- there are large gaps from the sequencing as compared to what was documented. Specifically, the sequenced biobrick was missing approximately 500bp
- the sequence was supposed to be 1326bp long. We have decided that it would cost too much to have the biobrick sequenced ourselves
- the gel is bad
- Wetlab
- OmpA-silicatein
- Curli
- Purchasing
- Human Practices
- Experimental Design/Characterization
- Project Planning
- Wiki
Created a timeline for the rest of the summer as seen below
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.
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
insert meeting minutes
Saturday, June 16
Removed the following parts from the incubator and placed in the refrigerator with parafilm wax
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.
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.
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
Today, we transformed pLac, TetR, terminator (BBa_B0015), RBS (BBa_B0034), pSB1A3, and pSB1C3.
The minipreps gave the following results
Thursday, June 21
Due to the low yield of our transformations and minipreps, we have now decided to troubleshoot our transformation procedures. To do this, we will be doing a transformation with Tarun, a graduate mentor to the team. We will be using Tarun's procedures and supplies. We will also have a control plate that will contain cells that had no DNA inserted. If these transformations succeed, then we will have narrowed the possibilities of what went wrong with our procedures. Tarun's transformation procedure can be found on the protocol page. We performed these test transformations with BBa_I13602 (CFP).
Upon closer inspection of the AIDA-1 biobrick (BBa_K257018), we have found the following problems
For the above reasons, we have decided to not use AIDA-1. After reevaluating the other adhesion options, we have decided to use Curli (BBa_342003). Due to its inconsistant sequencing, we have decided to have the part found in the kit sequenced ourselves.
Friday, June 22
Due to our increasing size, we have decided to divide into committees. These committees will help us divide the workload fairly and ensure that all tasks are being completed in a timely manner. The committees and their members are listed below. An asteric indicates the committee head.
*August
Haefa
*Rubeena
*Peter
*Peter
*Max
*Amanda
*Mrudula
Chris
Arthi
Rachel
Jim
*Namita
*Rachel
Sean (background)
We checked our test transformations from yesterday. None of the plates showed growth.
Monday, June 25
The foci of this week are to troubleshoot our transformations and research ways to characterize our constructs once they are assembled. August and Mrudula will be in lab troubleshooting. Amanda and Mrudula will be resarching ways to characterize a biofilm. Rachel will be researching ways to characterize a silica coat.
Friday, June 29
We went to Dow Agro, one of our sponsors, to give them an overview of our project. The powerpoint presentation we gave can be found Powerpoint. After we gave our presentation, we asked the Dow representative, Ryan, if he had any guidance with concern to Gibson assembly.
Saturday, June 30
Part of the team volunteered at the high school Jamboree. We saw many amazing teams with remarkable ideas! Congratulations to all the teams for their hard work over the past semester, and good luck as future iGEMers!
Sunday, July 1
We attempted another test transformation, this time using bought Excel 10β competent cells. We had both a negative and positive control. We also plated different concentrations of the final solution to see if we were over or under plating. We also used agar plates with ampicillin that Tarun made. We used store bought SOC. The part used was BBa_J04450.
Monday, July 2
The tranformations performed yesterday were successful. Both the positive control and the plaes with our part showed growth. The transformation efficiency was extremely high. We got single colonies on the plates with 50μL and overgrowth on the plates that had 150μL. We will only plate at 50μL from now on.
Due to time constraints, we have decided to synthesize both of our constructs. In parallel to characterization of the synthesized constructs, we are going to assemble both constructs using Gibson assembly. The assembles constructs will differ from the synthesized constructs in that the promoter will be a PoPs device and there will be a fluorescent protein on each. In this way, we can find if the addition of these two characterization parts will affect the functionality of our constructs.
The synthesized constructs were ordered from DNA 2.0. We are having two versions of the Curli construct made--one with a fluorescent reporter and one without. Pictures of the Gene Designer (DNA 2.0 software) renditions of the three constructs are given below.
We transformed Curli so that we can have it sequenced soon
Tuesday, July 3
We are now awaiting the delivery of our synthesized constructs. In preparation, we are researching the varying types of assays that we may be able to use. You can find information about the different assays we researched can be found here.
Silicatein-Alpha Characterization
We have decided to use scanning electron microscopy (SEM)< x-ray diffraction (XRD), and a live dead assay (specifically, the Baclight Bacterial Viability Kit from Invitrogen).
Further assays will be done once the two constructs are used within the same bacteria.
Monday, June 11
We attempted to optimize our primers using online guidlines and software.
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
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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
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Saturday, June 16
Removed the following parts from the incubator and placed in the refrigerator with parafilm wax
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.
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.
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
Today, we transformed pLac, TetR, terminator (BBa_B0015), RBS (BBa_B0034), pSB1A3, and pSB1C3.
The minipreps gave the following results
Thursday, June 21
Due to the low yield of our transformations and minipreps, we have now decided to troubleshoot our transformation procedures. To do this, we will be doing a transformation with Tarun, a graduate mentor to the team. We will be using Tarun's procedures and supplies. We will also have a control plate that will contain cells that had no DNA inserted. If these transformations succeed, then we will have narrowed the possibilities of what went wrong with our procedures. Tarun's transformation procedure can be found on the protocol page. We performed these test transformations with BBa_I13602 (CFP).
Upon closer inspection of the AIDA-1 biobrick (BBa_K257018), we have found the following problemsFriday, June 22
Due to our increasing size, we have decided to divide into committees. These committees will help us divide the workload fairly and ensure that all tasks are being completed in a timely manner. The committees and their members are listed below. An asteric indicates the committee head.
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*August
Haefa
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*Rubeena
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*Peter
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*Peter
*Max
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*Amanda
*Mrudula
Chris
Arthi
Rachel
Jim
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*Namita
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*Rachel
Sean (background)