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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
