Team:Davidson-MissouriW/Notebook

From 2010.igem.org

(Difference between revisions)
(Notebook)
(Notebook)
Line 305: Line 305:
<li>Added slider feature to the simulation program
<li>Added slider feature to the simulation program
<li>Continued work on the theoretical probability model for Construct A of size 3
<li>Continued work on the theoretical probability model for Construct A of size 3
 +
<li>Tested “cre construct” from 2009 iGem kit plate
</ul>
</ul>
<br>
<br>
Line 314: Line 315:
<li>Added custom prefixes and suffixes to the Oligator
<li>Added custom prefixes and suffixes to the Oligator
<li>Found a general equation for finding theoretical probabilities for Construct A of size 3
<li>Found a general equation for finding theoretical probabilities for Construct A of size 3
 +
<li>Sent off “floxed sites for sequencing”
</ul>
</ul>
<br>
<br>
Line 323: Line 325:
<li>Began analyzing MW optimization code
<li>Began analyzing MW optimization code
<li>Finished theoretical probabilities for Construct A of size 3
<li>Finished theoretical probabilities for Construct A of size 3
 +
<li>Cre construct from 2009 plate was incorrect as well
</ul>
</ul>
<br>
<br>
Line 332: Line 335:
<li>Worked on a separate program using the CAI equation
<li>Worked on a separate program using the CAI equation
<li>Began working on theoretical probabilities for Construct A of size 4
<li>Began working on theoretical probabilities for Construct A of size 4
 +
<li>Ligated more “floxed constructs”
</ul>
</ul>
<br>
<br>
Line 341: Line 345:
<li>Added changes to the optimizer code
<li>Added changes to the optimizer code
<li>Researched how to solve a system of recursive relations into closed form
<li>Researched how to solve a system of recursive relations into closed form
 +
<li>Decided to ligate cre into pBad+RBS and pLac+RBS
</ul>
</ul>
<br>
<br>
Line 350: Line 355:
<li>Fixed the error in the CAI optimization program
<li>Fixed the error in the CAI optimization program
<li>Continued analyzing systems of recursive relations
<li>Continued analyzing systems of recursive relations
 +
<li>Ligated pBad+RBS+Cre and pLac+RBS+Cre
</ul>
</ul>
<br>
<br>
Line 358: Line 364:
<li>Adapted the Oligator page for the Optimization program, now called the Optimus
<li>Adapted the Oligator page for the Optimization program, now called the Optimus
<li>Made progress on solving the system of recursive relations that we have
<li>Made progress on solving the system of recursive relations that we have
 +
<li>Performed IPTG and tet experiments
 +
<li>PCR screened both “cre constructs”
 +
<li>pBad+RBS+Cre ligation unsuccesful
</ul>
</ul>
<br>
<br>
Line 367: Line 376:
<li>Started to link the ruby program on the webpage
<li>Started to link the ruby program on the webpage
<li>Finished theoretical probabilities for Construct A of size 4
<li>Finished theoretical probabilities for Construct A of size 4
 +
<li>"Floxed constructs" ligated into pLac+RBS+Cre
</ul>
</ul>
<br>'''July 22, 2010'''
<br>'''July 22, 2010'''
Line 375: Line 385:
<li>Linked the Optimus on the webpage
<li>Linked the Optimus on the webpage
<li>Started theoretical probabilities for Construct A of size 5
<li>Started theoretical probabilities for Construct A of size 5
 +
<li>Cre was religated into pBad+RBS
</ul>
</ul>
<br>
<br>
Line 384: Line 395:
<li>Worked on the tutorial for the Optimus and added various features
<li>Worked on the tutorial for the Optimus and added various features
<li>Worked on a Matlab tool based on the pancake problem that can find the theoretical probabilities of different modules in different positions
<li>Worked on a Matlab tool based on the pancake problem that can find the theoretical probabilities of different modules in different positions
 +
<li>Last of the “floxed constructs” were ligated
</ul>
</ul>
<br>
<br>

Revision as of 16:08, 27 July 2010

Notebook

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


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



    July 27, 2010


    • Worked on team wiki.



    July 28, 2010


    • n/a
    • n/a



    July 30, 2010


    • n/a
    • n/a