Team:Purdue/Notebook

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== Protocols ==

Revision as of 14:26, 18 June 2012

Home Team Official Team Profile Project Parts Submitted to the Registry Modeling Notebook Human Practices Safety Attributions

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.

Contents

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

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?
  • Diagram of complete device was shown


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 Porterfielt 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)
  • Protocol: 
 1) Add 50μL of DH5α to 1μL of DNA
 2) Sit solution on ice for 20-30 minutes
 3) Heat shock the solution at 42°C for 45 seconds
 4) Return the solution to ic for 2 minutes
 5) Add 200μL of SOC
 6) Shake at 37°C and 200rpm for 1 hour
 7) Plate 100-150μL of samples onto agar dishes
  • 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

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
      • Complet 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.
    • [http://bioweb.uwlax.edu/genweb/molecular/seq_anal/primer_design/primer_design.htm| Here] is a website that details primer design.
    • We used a [http://www.neb.com/nebecomm/tech_reference/TmCalc/Default.asp| Tm calclator] and an [http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/| 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.
  • 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)

Thursday, June 14

  • Rachel had a demonstration on how to make silica. The procedure is shown below. 
    1) Obtain 14.4g of nanopure water
    2) Obtain 50µL of .04M HCL and combine it with the water. Put this solution in an ice bath
    3) Obtain 7.6g of tetramethyl orthosilicate (TMOS) in a separate container
    4) Combine the TMOS and HCL/water 
    5) Immediately begin the vortex the solution. The solution will be cloudy while the reaction is taking place.
       Once the solution is clear again, the reaction is finished. This will take approximately 5 minutes
    6) When the reaction is finished, let the solution set for 1-2 minutes
    7) Use a rotary evaporator at 47ºC to remove the methane from the solution. This should take 2-3 minutes. The
       final
       solution volume should be approximately 13mL
    8) If the silica is to be used immediately, put it on ice. It can be stored for up to a week if it is
       refrigerated. 
    9) Filter the silica before use
  • 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_

Protocols

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