# Team:Davidson-MissouriW/Notebook

### From 2010.igem.org

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<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> | ||

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<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> | ||

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<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> | ||

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<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> | ||

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<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> | ||

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<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> | ||

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<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''' | ||

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<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> | ||

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<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

**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