Team:Davidson-MissouriW/Notebook

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Revision as of 16:21, 28 July 2010

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

July 28, 2010

July 29, 2010

July 30, 2010

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