Team:Davidson-MissouriW/Notebook

From 2010.igem.org

Notebook

May 25, 2010 to May 29, 2010


  • Davidson traveled to Missouri Western and further discussed project ideas


June 1, 2010


  • Tested to find lethal concentration of Tet in plated and liquid mediums
  • Verified RCBS with our sequences and researched codon optimization methods
  • Analyzed probabilities for constructs A and B
  • Learned lab protocols and organized lab
  • Created list of needed parts and parts we need


June 2, 2010


  • Mini prepped, diagnostic RP digest, preparative digest, ligation, pBAD+RBS-RFP
  • Researched different ways to optimize a gene
  • Began simulation for Constructs A and B
  • Learned miniprep and digestion protocols
  • Learned how to document gels


June 3, 2010


  • Mini prepped, diagnostic RP digest, preparative digest, ligation, pBAD+RBS-RFP
  • Researched restriction sites and created proposal for gene optimization
  • Continued work on simulation in Java and Matlab
  • Learned about biobrick restriction sites and sticky ends
  • Ordered lox oligos


June 4, 2010


  • Mini prepped RBS-TetA-TT
  • RX Fragment digestion for RBS-TetA-TT
  • Analyzed the optimization of tetA sequence and researched properties of Lox sites
  • Debugged Simulations
  • Sent off parts for sequencing
  • Learned the transformation protocol


June 7, 2010


  • Ligate and plasmid check for pSB1A2 or pSB1AK3
  • Began working to improve the Lancelator and created histograms for simulations
  • Gel purified vector for lox ligations
  • Divided up constructs to be ligated


June 8, 2010


  • Double digest on pBAD-RBS-RFP to make an RS fragment insert
  • Worked on a Perl program that check for Bio Brick Restriction Sites
  • Analyzed sequencing results using ApE program
  • BOUGHT HAWAIIAN SHIRTS!!!!!


June 9, 2010


  • EcoRI and PstI digest on pLac and pBad colonies
  • Arabinose and IPTG concentration experiments started
  • Continued work on the Lancelator program and began working on a different simulation for Construct C and D
  • Annealed lox oligos together
  • Gel purified pBad+RBS+Cre (“Cre construct” from 2010 igem kit plate)


June 10, 2010


  • Picked 14 colonies from pLac-RBS-RFP and pLac-RBS-RFP-RBS plates
  • Started inducer curve experiments
  • Linked Lancelator code to the website and continued simulations
  • Performed ligations involving lox sites


June 11, 2010


  • Ligated S03736 into pSB1A7 vector
  • EcoRI/PstI digest on pLac-RBS-RFP-RBS colonies
  • Added new features to the Lancelator and worked on numerically representing Construct C for the Matlab simulation
  • Performed colony PCR screening for the first ligations


June 14, 2010


  • 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
  • Tested “cre construct” from 2009 iGem kit plate


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
  • Sent off “floxed sites for sequencing”


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
  • Cre construct from 2009 plate was incorrect as well


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
  • Ligated more “floxed constructs”


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
  • Decided to ligate cre into pBad+RBS and pLac+RBS


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
  • Ligated pBad+RBS+Cre and pLac+RBS+Cre


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
  • Performed IPTG and tet experiments
  • PCR screened both “cre constructs”
  • pBad+RBS+Cre ligation unsuccesful


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
  • "Floxed constructs" ligated into pLac+RBS+Cre


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
  • Cre was religated into pBad+RBS



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
  • Last of the “floxed constructs” were ligated



July 26, 2010


  • Missouri traveled to Davidson to work on wiki and presentation materials
  • The two campuses reunite and share recent findings



July 27, 2010


  • Began work on the team wiki



October 25, 2010


  • Finalizing team wiki


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