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From 2011.igem.org

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Notebook

Week of 5/23/2011

Design Team: Started collecting sequences of relevant fluorescent proteins and entering them into GenoCAD. Learned about attribute grammars.

Fabrication Team: Lab Training.

Characterization Team: Started looking at model, ways of analyzing data that we'll eventually get, and fitting model to it. Started training on microscope, cell culture. Started learning Matlab for GenoSIGHT changes.

Week of 5/30/2011

Design Team: Created first draft of attribute grammar. Collected more fluorescent proteins for GenoCAD. Started looking at modeling aspect of project - explored modeling software that can handle SBML (Systems Biology Markup Language) input - Matlb Simbiology looks nice.

Fabrication Team: We were split into two teams to work autonomously on Miniprep procedure for plasmid DNA extraction and SalI and EcoRI restriction digests of mCherry and pDSRed.

Characterization Team: Started looking at parameter estimation - methods of getting maturation and degradation rates from fluorescence over time data. More Matlab training. More microscope training, in preparation for real experiments once fab team finishes transforming some cells.

Week of 6/6/2011

Design Team: Small changes to grammar (fixing stop codon issues) and big changes to grammar (changing how parts are ordered and thus how GenoCAD constructs the sequence of a construct we make). Looked further into modeling, how our attribute grammar will connect to that. Collected more fluorescent proteins, started to look into degradation tags and linker sequences for fusion proteins.

Fabrication Team: We were split into two teams to work autonomously on Miniprep procedure for plasmid DNA extraction and SalI and EcoRI restriction digests of mCherry and pDSRed.

Characterization Team: More work on parameter estimation, common algorithms for doing this. More Matlab training, started to modify GenoSIGHT by adding Time Calculator to GUI.

Week of 6/13/2011

Design Team: Started working on Prolog compiler which will take a construct from GenoCAD and use it to write a Java file, which will in turn write an SBML file for modeling (this is a good way to do it, we promise). Carefully documented gene sequences we've found so far. Looked more into best degradation tags to use. Started learning how to verify sequencing results so we can look at sequencing data from our constructs made by the fab team.

Fabrication Team: We were split into two teams to work autonomously on Miniprep procedure for plasmid DNA extraction and SalI and EcoRI restriction digests of mCherry and pDSRed.

Characterization Team: Started imaging actual cells. More work on parameter estimation problem and comparing algorithms. Started looking for new parts in Registry. More changes to GenoSIGHT GUI.

Week of 6/20/2011

Design Team: More work on Prolog compiler (long and frustrating work). Lots of troubleshooting with the fab team picking a better plasmid to use in E. coli Verified that our grammar implementation in GenoCAD is producing accurate DNA sequences for constructs.

Fabrication Team: We compared the differences in protocol between the Yeast pYES2 vector and E. coli pDsRed vector and updates on Codon Optimization.

Characterization Team: More imaging of cells from fab team. More work on parameter estimation. Started looking at adaptive imaging and timing. Troubleshooting with Design Team.

Week of 6/27/2011

Design Team: Added linker sequences and degradation tags to GenoCAD, along with plasmids. Got compiler working - we can now make a construct in GenoCAD and generate a valid SBML file, which can be simulated in Matlab, etc. Started writing iGEM safety proposal and project description. Started adapting grammar to make a bigger variety of constructs.

Fabrication Team: We worked on mCherry and mCitrine from pRSETb into pYES2 for yeast expression, and Biobrick ECFP (BBa_E0020) is being used as a test protein to validate our chosen backbone vector (pSB1AK3 with part J04500). We also worked on converting our existing fluorescent proteins into Biobrick parts in order to further test pSB1AK3.

Characterization Team: More imaging and updates to GenoSIGHT GUI. Starting to test parameter estimation against simulated data.

Week of 7/4/2011

Design Team: Finished safety proposal and project description. Looked into software to simulate our SBML files. Major changes to grammar to be able to make more types of constructs. Added N-terminal degradation tags to grammar and GenoCAD.

Fabrication Team: Hayley worked on mCitrine insert in pYES2 backbone and mCherry (E2060) insert in pSB1AK3 backbone with Lac promotor and RBS binding site (J04500). Loran worked on mCherry insert in pYES2 backbone and Plasmid Prep BioBrick mCherry and CFP (E0020) inserts. Mandy worked on CFP insert in pSB1AK3 backbone and mCherry insert in PYES2 backbone. Martha worked on GFPmut3b in pSB1AK3 backbone. Swetha worked on mCitrine insert in pYES2 backbone and Degradation Tags Primer Design.

Characterization Team: More imaging and updates to GenoSIGHT GUI. Testing parameter estimation against simulated data. Photobleaching issues. Work on adaptive imaging and timing.

Week of 7/11/2011

Design Team: Finished safety proposal and project description again. Started verifying sequences of constructs made by fab team.

Fabrication Team: Loran completed mCherry-pYES2 and started cloning non-Biobrick AcGFP-pYES2 and Biobrick AcGFP-pSB1AK3. Swetha completed mCitrine-pYES2 and started cloning Biobrick CFP-pYES2 and Biobrick GFPmut3b-pYES2. Mandy completed CFP-pSB1AK3 and mCherry-pYES2 and started cloning Biobrick mCherry-pYES2, Biobrick mCherry-pSB1AK3, and Biobrick GFPmut3b-pSB1AK3. Hayley completed mCitrine-pYES2 and started cloning Biobrick mCherry-pSB1AK3 and non-Biobrick mCitrine-pSB1AK3. Martha started cloning Biobrick GFPmut3b-pSB1AK3.

Characterization Team: Lots of imaging - lots of constructs coming from fab team now. More work on getting parameter estimation to work efficiently and as expected. Adaptive imaging for GenoSIGHT.

Week of 7/18/2011

Design Team: Lots of sequence verification. Started writing up details of our procedures and methods (materials used, manufacturers, etc). Started editing wiki. Explored ways of representing our grammar (just a list of rules) graphically, for easier interpetation.

Fabrication Team: We worked on the mCitrine-J04500, AcGFP-pYES2, GFPmut3b-pYES2, and CFP-pYES2 constructs.

Characterization Team: Lots more imaging. More photobleaching troubleshooting. More work on getting parameter estimation to work efficiently and as expected. Adaptive imaging for GenoSIGHT.

Week of 7/25/2011

Design Team: Started to work on presentation for iGEM, more work on wiki. Continued talking to other teams to write up our methods and procedures. Major changes to database where our grammar and parts are stored to accomodate attributes better.

Fabrication Team: We have designed new primers for constructs with tags, which need to be tested over a range of PCR temperatures to find the optimum. The mCitrine insert with Sul20C tag in J04500 backbone, mCitrine insert with LVA tag in J04500 Backbone, acGFP insert with LVA tag in J04500 backbone, adn GFPmut3B (E0040) with PEST Tag in pYES2 Backbone have been completed.

Characterization Team: Lots of imaging, more troubleshooting of equipment failures. Parameter estimation working pretty nicely now. Adaptive imaging working successfully on microscope.

Week of 8/1/2011

Design Team: Lots of work on presentation for iGEM. Continued talking to other teams, writing up our methods and procedures. Mark had to leave for the summer because of health issues :(

Fabrication Team: Helped with presentation, poster, and wiki.

Characterization Team: Helped with presentation, lots and lots of imaging - trying to finish imaging everything fab team has made. Getting actual parameters for data we collected!

Week of 8/8/2011

Design Team: Lots and lots of work on presentation. Final wrap-up, careful documentation of what we did all summer, etc.

Fabrication Team: Helped with presentation, poster, and wiki.

Characterization Team: Continued helping with presentation. Got some more parameters for our fluorescent proteins. Lots of imaging to finish getting data on all the constructs we made.