iGEM 2010 — Team Davidson-MissouriW

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# Team:Davidson-MissouriW/Notebook

### From 2010.igem.org

* [Home](https://2010.igem.org/Team%3ADavidson-MissouriW)
* [Team](https://2010.igem.org/Team%3ADavidson-MissouriW/Team)
  + [Overview](https://2010.igem.org/Team%3ADavidson-MissouriW/TeamBio)
  + [Advisors](https://2010.igem.org/Team%3ADavidson-MissouriW/Team#Advisors)
  + [Students](https://2010.igem.org/Team%3ADavidson-MissouriW/Team#Students)
* [Project](https://2010.igem.org/Team%3ADavidson-MissouriW/Project)
  + [Abstract](https://2010.igem.org/Team%3ADavidson-MissouriW/Project#Abstract)
  + [Introduction](https://2010.igem.org/Team%3ADavidson-MissouriW/Project#Introduction)
  + [Optimizing Codons](https://2010.igem.org/Team%3ADavidson-MissouriW/OptimizingCodons)
  + [Characterizing Cre/Lox](https://2010.igem.org/Team%3ADavidson-MissouriW/CreLox)
  + [Measuring Gene Expression](https://2010.igem.org/Team%3ADavidson-MissouriW/MeasuringExpression)
  + [Summary & Outlook](https://2010.igem.org/Team%3ADavidson-MissouriW/Project#Summary and Outlook)
  + [References](https://2010.igem.org/Team%3ADavidson-MissouriW/Project#References)
* [Parts](https://2010.igem.org/Team%3ADavidson-MissouriW/Parts)
* [Tools](https://2010.igem.org/Team%3ADavidson-MissouriW/Tools)
  + [Veripart](https://2010.igem.org/Team%3ADavidson-MissouriW/Tools#Veripart)
  + [Oligator](https://2010.igem.org/Team%3ADavidson-MissouriW/Tools#Oligator)
  + [Optimus](https://2010.igem.org/Team%3ADavidson-MissouriW/Tools#Optimoose)
  + [SimuLox](https://2010.igem.org/Team%3ADavidson-MissouriW/Tools#Simulator)
* [Human Practices](https://2010.igem.org/Team%3ADavidson-MissouriW/HumanPractices)
* [Notebook](https://2010.igem.org/Team%3ADavidson-MissouriW/Notebook)
* [Safety](https://2010.igem.org/Team%3ADavidson-MissouriW/Safety)
* [Acknowledgements](https://2010.igem.org/Team%3ADavidson-MissouriW/Sponsors)

## 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**

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* 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**

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* 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**

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* 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**

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* Began work on the team wiki

**October 25, 2010**

---

* Finalizing team wiki

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