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From 2012.igem.org

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 - we identified the reducible elements of our system, detailed an outline of the project to the SURF students (e.g. 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.

• Tet repressor: 79.8ng/μL
• PoPs receiver: 149.6ng/μL
• Lac promotor and CFP generator: 46.2ng/μL
• Tet promotor wih GFP: 22.9ng/μL

These concentrations are too low to use in any assembly method; therfore, we will need to attempt transformations and minipreps again before doing any assembly method.

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)

Here is a powerpoint summarizing our team meeting.

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

Here is a powerpoint summarizing our team meeting.

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

1. RBS: 14.8ng/μL
2. TetR: 5.0ng/μL
3. CFP: 9.0ng/μL
4. PoPs: 14.5ng/μL
5. 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 here. 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.

Here is a powerpoint summarizing our team meeting.

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.

Tuesday, June 26

Here is a powerpoint summarizing our team meeting.

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. Pictures of the Gene Designer (DNA 2.0 software) renditions of the three constructs are given below.

• Silicatein construct
• Curli

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.

Thursday, July 5

Amanda did research on possible competitors for our product. This information can be used for quality control of our product. The competitors Amanda found are listed below.

• Hydro Engineering - produces "Hydrokleen", which is a bioreactor filtration system that would generally be used in industrial plants and acts as a water recycler
• Seven Trent Services - produces Tetra Amphidrone fixed sequencing Batch Biological Filter, a waste water treatment process that uses a biological filter to remove various pollutants
• Kinetics - this company makes a range of water treatment processes and machines that can be used in industry as well as consumer markets
• ABEC - produces bioreactors, but could not find more detail into what kind

Friday, July 6

Here is a powerpoint summarizing our team meeting.

Sunday, July 8

Research was done on growing a static biofilm. "Growing and Analyzing Static Biofilms" was a very helpful article.

Monday, July 9

We performed a Growth Rate Assay on the NEB Beta and the DH5 alpha in order to know when the bacteria reach the different stages of growth. This knowledge will be essential for growing static biofilms as well as doing many assays. This is a test to figure out what is happening. The results of this assay can be found here.

Tuesday, July 10

We performed the Growth Rate Assay on XL-10 Gold. The results of the assay can be found here.

Wednesday, July 11

Chris T created primers for a test Gibson reaction. The test construct will be a the plasmid pGA3K3, PoPs device (BBa_F2620), an RFP with RBS(BBa_K093005), and a double terminator (BBa_B0014). The primers can be viewed in this document . We ordered the primers from Integrated DNA Technologies.

Friday, July 13

We performed a Static Biofilm Assay.

Thursday, July 19

We attempted to do PCR for the parts for which Chris made primers. To prove that the PCR was successful, we ran aliquotes of the reaction products through a gel. An image of the gel can be seen here. No bands can be seen for the aliquotes; therfore, we concluded that the PCR did not work. We believe that there was a problem with the cycler; therefore, we will be trying the procedure again with a different machine.
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