Team:Davidson-MissouriW/Notebook

• EcoRI/PstI diagnostic digest performed on pBad-RBS-RFP colonies.
• Inducer curve experiment started on pBad and pLac constructs.
• Debugged Lancelator and finished numerical representation for the simulation
• Size verified lox forward and lox reverse sites
• Ligated “cre construct” into low, medium, and high copy kanamycin resistant vectors

June 15, 2010

• Ligated pLac-RBS-RFP + RBS-TetA.
• IPTG experiment started on pLacI-RBS-RFP
• Worked on the color schemes for the new Oligator
• Created a new construct that might be helpful for the biologists
• Planning for Amp+Tet experiments
• First “cre construct” ligations failed

June 16, 2010

• EcoRI/PstI diagnostic digest performed on pLac-RBS-RFP in pSB1A7 vector.
• IPTG experiment started on plates with pLac and pLacI constructs.
• Began working on a Java code that will run simulations for all the constructs
• Researched on NP-Complete problems
• Religated “cre construct”
• Lox sites sent off for sequencing

June 17, 2010

• Flourimeter data collected from IPTG experiment.
• Ligated pLacI-RBS-RFP + RBS-TetA.
• Worked on graphics of the Java program
• Studied specific NP-Complete problems and brainstormed ways to incorporate them in our project
• Lox sites were frozen down and entered in GCAT-alog
• Agar plates were prepared for Amp+Tet experiments

June 18, 2010

• Picked 4 colonies from pLacI-RBS-RFP-RBS-TetA plates.
• IPTG experiment started using newly created IPTG.
• Debugged the Java program
• Created a new page for the GCAT server
• Planned kanamycin experiments

June 21, 2010

• Digested TetA with EcoRI/NheI.
• Annealed oligos for optimized and deoptimized TetA segment 1.
• Improved the Java program
• Began categorizing the knapsack problem into subparts
• Successfully ligated ptet+different lox sites
• Planned different combinations of variant lox sites

June 22, 2010

• Ligated annealed TetA oligos into digested TetA.
• Inducer curve experiment started on pLac and pLacI constructs.
• Added buttons to the java program
• Brainstormed biological implications in different NP-Complete problems
• Confirmed successful ligations of lox sites from sequencing results
• First pilot test for Amp+Tet experiments

June 23, 2010

• Preparative NheI/BamHI digest performed on TetA.
• Preparative EcoRI/NheI digest performed on TetA.
• Restructuring and reorganizing the Java program
• Reviewed numerical algorithms and its relation to NP-Complete problems
• Researched data on possible transcription terminator in Tet A
• Failure of cre experiments

June 24, 2010

• Annealed optimized and deoptimized segment 1 TetA ligated into vector.
• Annealed optimized and deoptimized segment 2 TetA ligatied into vector.
• Added weight characteristic to the fluorescent proteins in the Java code
• Continued exploration of NP-Complete problems
• Ligations to construct tet constructs

June 27, 2010

• Inducer curve experiment started on pLac and pLacI constructs.
• Picked colonies from TetA segment 1 & 2 optimized & deoptimized plates.
• Added histograms to the Java Code
• Researched and analyzed the effectiveness of Cre
• Size verification of ptet+variant lox sites

June 28, 2010

• Added the option for the user to input the weights of the various modules and began work on custom construct builder
• Picked colonies from TetA segment 1 & 2 optimized & deoptimized plates.
• Planned to ligate pBad+RBS+Cre in ampicillin resistant vectors
• Researched plasmid partitioning

June 29, 2010

• Screened segment 1 optimized and deoptimized candidate clones using RFLP
• Screened segment 2 optimized and deoptimized candidate clones using RFLP
• Finished the simulation program and exported it
• Explored the set covering problem
• Researched applications of the knapsack problem in cryptography
• Ligated ptet+LoxP+RBS+RFP

June 30, 2010

• Tet titration experiment performed on pLac and pLacI +RBS-RFP-RBS-TetA.
• Ligated TetA onto pLac and pLacI +RBS-RFP-RBS-TetA with no terminator.
• Some final work on the simulation program
• Discussed new ideas such as integer programming
• PCR screening of “RFP construct”
• Ptet+variant lox sites were frozen down

July 1, 2010

• Preparative BamHI/NheI digest performed on TetA.
• New experiment protocol developed for reduced vector background noise.
• Added terminators to the program
• Ran test trials of the program and analyzed its results
• Planned experiments for using Cre as “front” and “back” vector
• Ligated first “floxed” constructs

July 2, 2010

• Mini prepped, diagnostic RP digest, preparative digest, ligation, pBAD+RBS-RFP
• Continued work on the categorization of the knapsack problem
• Researched Instant Insanity problem
• PCR screening for “floxed constructs”

July 6, 2010

• Ligated S04446 and S04447 to J31007.
• Tet titration experiment performed on pLac and pLacI +RBS-RFP-RBS-TetA.
• Added direction of terminators in the simulation
• Researched cryptography application
• Size verified “floxed constructs”
• Digested “Cre construct” as front and back vector

July 7, 2010

• Ligated S04448 to RBS-TetA.
• Ligated S04449 to RBS-TetA.
• Fixed memory leak in the simulation program
• Researched integer programming
• Sent off “floxed constructs" for sequencing
• Ligated “floxed constructs” into pBad+RBS+Cre as “front” and “back” vector

July 8, 2010

• IPTG experiment started using pre-induced method.
• IPTG experiment started using varying levels of tetracycline.
• Worked on animation for the simulation and continued analysis of the results of the simulation
• Discussed integer programming as a group
• Data suggests something wrong with “cre construct”

July 9, 2010

• Picked optimized and deoptimized TetA colonies.
• Flourimeter data collected from IPTG experiment.
• Revised some features on the program
• Began working on theoretical probability models of the different constructs
• Further experiments with “cre construct”

July 10, 2010

• Used RFLP to screen for candidate TetA clones.
• Transformed various pLac and pLacI constructs into MG1655 cells.
• Experiments suggest pBad+RBS+Cre from iGem 2010 kit plate is incorrect

July 12, 2010

• Used RFLP to screen for candidate TetA clones.
• Transformed various pLac and pLacI constructs into MG1655 cells.
• Added slider feature to the simulation program
• Continued work on the theoretical probability model for Construct A of size 3

July 13, 2010

• Used RFLP to screen for candidate TetA clones.
• EcoRI/PstI diagnostic digest performed on K199150.
• Added custom prefixes and suffixes to the Oligator
• Found a general equation for finding theoretical probabilities for Construct A of size 3

July 14, 2010

• Ligated segment 2 optimized and deoptimzed TetA behind segment 1.
• IPTG experiment started on I715039-1 and I715039-2.
• Began analyzing MW optimization code
• Finished theoretical probabilities for Construct A of size 3

July 15, 2010

• Cell count data collected from IPTG experiments.
• Flourimeter data collected from IPTG experiments.
• Worked on a separate program using the CAI equation
• Began working on theoretical probabilities for Construct A of size 4

July 16, 2010

• Tet titration experiment started using pSB3T5 vector.
• Tet titration experiment started using optimized and deoptimized TetA.
• Added changes to the optimizer code
• Researched how to solve a system of recursive relations into closed form

July 19, 2010

• Cell count data collected from Tet titrations.
• Tet titration experiment started using optimized and deoptimized TetA.
• Fixed the error in the CAI optimization program
• Continued analyzing systems of recursive relations

July 20, 2010

• Flourimeter data collected on IPTG experiment.
• Adapted the Oligator page for the Optimization program, now called the Optimus
• Made progress on solving the system of recursive relations that we have

July 21, 2010

• IPTG experiment started on optimized and deoptmized TetA constructs.
• Passage I715039-1 and I715039-2 experiments started.
• Started to link the ruby program on the webpage
• Finished theoretical probabilities for Construct A of size 4

July 22, 2010

• IPTG experiment started using varying tetracycline concentrations.
• Cell count data collected on IPTG TetA experiment.
• Linked the Optimus on the webpage
• Started theoretical probabilities for Construct A of size 5

July 23, 2010

• Cell count data collected from IPTG experiment..
• Flourimeter data collected from IPTG experiment.
• Worked on the tutorial for the Optimus and added various features
• Worked on a Matlab tool based on the pancake problem that can find the theoretical probabilities of different modules in different positions

July 26, 2010

• Presented Missouri Western data to Davidson colleagues.
• Presented Davidson data to Missouri Western colleagues.

July 27, 2010

• Worked on team wiki.

July 28, 2010

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July 30, 2010

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