Team:RHIT/Notebook

From 2012.igem.org

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<h3><i>S. Cerevisiae</i></h3>
<h3><i>S. Cerevisiae</i></h3>
<p>BY4741 (ATCC #201388): <i>MATa his3-Δ0 leu2-Δ0 met15-Δ0 ura3-Δ0</i></p><br />
<p>BY4741 (ATCC #201388): <i>MATa his3-Δ0 leu2-Δ0 met15-Δ0 ura3-Δ0</i></p><br />
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<p>BY4742 (ATCC #201389): <i>MATα his3-Δ0 leu2-Δ0 lys2-Δ0 ura3-Δ0</i></p>
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<p>BY4742 (ATCC #201389): <i>MATα his3-Δ0 leu2-Δ0 lys2-Δ0 ura3-Δ0</i></p><br />
-
<p>Transformations were carried out on competent E.Coli using the procedure available on the <a href="http://www.neb.com/nebecomm/products/protocol119.asp">NEB website</a>.</p>
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<p>Transformations were carried out on competent E.Coli using the procedure available on the <a href="http://www.neb.com/nebecomm/products/protocol119.asp">NEB website</a>.</p><br />
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<p>DNA extraction from cells was performed using a Qiagen QIAprep Spin Miniprep Kit (Cat. no. 27104). Included procedure was followed.</p>
+
<p>DNA extraction from cells was performed using a Qiagen QIAprep Spin Miniprep Kit (Cat. no. 27104). Included procedure was followed.</p><br />
-
<p>Digestions were performed using EcoRI, XbaI, SpeI and PstI using recommended NEB procedure.</p>
+
<p>Digestions were performed using EcoRI, XbaI, SpeI and PstI using recommended NEB procedure.</p><br />
-
<p>Gel extraction of digested DNA was performed using a Qiagen QIAEX II Gel Extraction Kit (Cat. no. 20021). Included procedure was followed.</p>
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<p>Gel extraction of digested DNA was performed using a Qiagen QIAEX II Gel Extraction Kit (Cat. no. 20021). Included procedure was followed.</p><br />
-
<p>Ligations were performed following procedure available on the <a href="http://www.neb.com/nebecomm/products/protocol658.asp">NEB website</a>.  Total mass of DNA for ligations was 100ng with a 1:3 to 1:4 ratio of vector to insert.  </p>
+
<p>Ligations were performed following procedure available on the <a href="http://www.neb.com/nebecomm/products/protocol658.asp">NEB website</a>.  Total mass of DNA for ligations was 100ng with a 1:3 to 1:4 ratio of vector to insert.  </p><br />
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Revision as of 15:33, 3 October 2012

Home
Team
Project
BioBricks
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Notebook
Outreach
Daily Log
Lab Notebook
Lab Protocols
Safety

Week 1

  • 6/4/2012: First round of project planning, lab clean-up, general outline for summer.

  • 6/5/2012: Second round of project planning, lab clean-up, research into Johns Hopkins 2008 summer project.

  • 6/6/2012: Third and final round of project planning, final lab clean-up, research into the pigment pathways and reporter genes, analysis of Cambridge project.

  • 6/7/2012: Broke down Johns Hopkins 2008 project, results in wiki, found pigments for A and a, found possible fluorescence colors, found possible proteins.

  • 6/8/2012: Research on Promoters, updated Next Actions.

Week 2

  • 6/11/2012: Identified a positive feedback loop that's been done in yeast before and a possible selection scheme, started plasmid mapping, set up server for wiki, began wiki design and looked at color schemes, block-diagrammed positive feedback loop, ate a whole tray of brownies.

  • 6/12/2012: Found gene sequences, continued work on graphic for top of Wiki, looked for selection schemes, shot down selection schemes.

  • 6/13/2012: Fluorescence construct, research on Gibson assembly and gateway vectors, updated wiki to make it more functional, talked about selection schemes, obtained 2 posters, tooks some pics.

  • 6/14/2012: Preparation for Journal Club, lunch with IRC and Math REU people, checked for sequences for plasmid construction.

  • 6/15/2012: Used Gene Designer to do primilary sequencing of plasmid, research on spacing of plasmid.

Week 3

  • 6/18/2012: Optimized DNA sequences, found all of the filler DNA.

  • 6/19/2012: Talked with Dr. Almaas, started web design, took pictures with storm troopers, team movie night.

  • 6/20/2012: Web design, learned HTML.

  • 6/21/2012: Web design, preliminary website up, team lunch, IRC meeting.

  • 6/22/2012: Individual work day.

Week 4

  • 6/25/2012: Group discussion, work on school website, organization of collected information.

  • 6/26/2012: Obtained shirts, found and organized all sequences, started to verify sequences, school page is 85% done.

  • 6/27/2012: Changes to DNA sequences, worked on finalization of sequence, looked at a 3D modeling program, contacted original DNA company about sequencing.

  • 6/28/2012: Worked on finalizing the sequence, identified materials needed for Gibson assembly, went to IRC presentation on grad. school/UC from Rose Undergraduate.

  • 6/29/2012: Individual work day.

Team Break Week

Week 5

  • 7/9/2012: Got back from break. Learned DNA had not shipped yet. :( Worked on wiki.

  • 7/10/2012: Worked on wiki.

  • 7/11/2012: Received edited version of what original DNA company will do to our DNA sequences. We compared them to the originals and prepared questions for original DNA company.

  • 7/12/2012: Talked to original DNA company about questions.

  • 7/13/2012: Individual work day.

Week 6

  • 7/16/2012: Decided to use GeneArt for sequences instead of original DNA company.

  • 7/18/2012: Brainstormed ideas for side projects while waiting for DNA shipment.

  • 7/19/2012: Continued brainstorm, expanding into Human Practices.

  • 7/20/2012: Created teams: Wiki, Presentation, Poster, and Maya.

  • 7/21/2012: Met in teams to plan and brainstorm. Added Team Board Game.

Week 7

  • 7/23/2012: Devon and Kristen began planning the THCM exhibit.

  • 7/24/2012: Continued work in Teams. Began intial research into pathway for modeling.

  • 7/25/2012: Continued work in Teams; added Team Modeling.

  • 7/26/2012: Continued work in Teams.

  • 7/27/2012: Continued work in Teams, lunch with IRC people

Week 8

  • 7/30/2012: Ironed out wiki formatting, began adding content, continued Maya animation.

  • 7/31/2012: Lab work, continued adding content to wiki.

  • 8/1/2012: Lab work, added more content to wiki, got update on additional computer for Maya animation, made significant progress on math model, Rose-Hulman team page went live.

  • 8/2/2012: Lab work, continued work in Teams.

  • 8/3/2012: Lab work, continued work in Teams.

Week 9

  • 8/6/2012: Canoe trip with IRC members, lab work.

  • 8/7/2012: Lab work, focus on modeling information for NTNU, continued work in Teams.

  • 8/8/2012: Lab work, Second Skype meeting with NTNU, conclusion and review of Maya animation.

  • 8/9/2012: Lab work, continued work in Teams.

  • 8/10/2012: Lab work, continued work in Teams.

Week 10

  • 8/13/2012: Lab work, continued work in Teams.

  • 8/14/2012: Lab work, continued work in Teams.

  • 8/15/2012: Lab work, continued work in Teams.

  • 8/16/2012: Lab work, plans made for continuing into the school year.

  • 8/17/2012: Cleaned workspace, finalized plans for continuation.

July 31, 2012

Made media: YPD, L-AMP, CSM, and CSM-His


Members: Ben, Bobby, Alex, Kristen, Devon, Dr. A.

August 1, 2012

Streaked out plasmid-bearing E. coli strains (pRS416 & pRS413) and yeast strains
Members: Ben, Bobby, Alex, Kristen, Devon, Dr. A.

August 2, 2012

Checked for E. coli colonies: success. Made liquid L-AMP media, innoculated small cultures.
Members: Ben, Bobby, Alex, Kristen, Devon, Dr. A.

August 3, 2012

Qiagen minipreps
Members: Ben, Bobby, Alex, Kristen, Devon, Dr. Dave.

August 6, 2012

Made stock solutions for transformation procedures
Members: Adam, Kristen, Dr. A

August 7, 2012

Transformed E. coli with our red construct
Members: Bobby, Adam, Devon, Kristen, Ben, Dr. A

August 8, 2012

Inoculated liquid media with transformed E.Coli
Members: Bobby, Adam

August 9, 2012

Ran Mini-Preps of pRS413, pRS416, and Red construct bearing E. Coli, stored extracted DNA
Members: Bobby, Adam

August 10, 2012

Ran quick gel of extracted DNA
Members: Bobby, Adam

August 11, 2012

Digested extracted DNA, isolated pRS413 & pRS416 fragments from gel
Members: Bobby, Adam

August 12, 2012

Isolated red construct fragments on gel, poured chloramphenicol plates
Members: Bobby, Adam

August 13, 2012

Digested extracted DNA, isolated pRS413, pRS416, and Red Construct from gel, Transformed E. Coli with NTNU construct
Members: Bobby, Adam, Dr. A

August 14, 2012

Ligated pRS413 and red construct, transformed E. Coli with ligated plasmid, inoculated liquid media with NTNU E. Coli
Members: Bobby, Adam

August 15, 2012

Mini-Preps of NTNU construct, confirmed presence on gel, Digested pRS413, pRS416, and Red Construct, poured ampicillin plates
Members: Bobby, Adam

August 16, 2012

Isolated pRS413, pRS416, and Red Construct from gel, began storing strains, DNA, and equipment
Members: Bobby, Adam

August 17, 2012

Ran digestion of pRS413, pRS416, and Red Construct, stored remaining strains, DNA, and equipment
Members: Bobby, Adam, Dr. A

August 28, 2012

Transformed E.Coli with BBa_E0422 reporter
Members: Bobby, Adam, Dr. A

August 29, 2012

Digested and isolated Red Construct and pRS413 from gel, Inoculated liquid media with BBa_E0422 reporter
Members: Adam

August 30, 2012

Digested pRS413 and Red Construct, Isolated from gel and Ligated, Transformed E. Coli.
Members: Adam

August 31, 2012

Digested pRS413 and Red Construct
Members: Bobby, Adam, Dr. A

September 1, 2012

Started cultures of pRS413 and the Red construct form frozen cultures
Members: Adam

September 2, 2012

Inoculated liquid media with colonies from plates of pRS413 and Red Construct strains
Members: Adam

September 3, 2012

Ran mini-preps of liquid colonies
Members: Adam

September 4, 2012

Ran diagnostic gel to check amount of DNA extracted
Members: Adam

September 5, 2012

Purified and concentrated DNA samples
Members: Adam

September 6, 2012

Ran diagnostic gel to check for purification success
Members: Adam

September 7, 2012

Ran BioBrick Assembly protocol on NTNU lld promoter and BBa_E0422, Transformed E.Coli with ligation mixture
Members: Adam, Dr. A

September 8, 2012

Inoculated liquid cultures with transformed E.Coli
Members: Adam

September 10, 2012

Digested and isolated Red Construct and pRS413 from gel, Ran mini-preps of liquid cultures
Members: Adam

September 11, 2012

Ran gel of lld/0422 hybrid, Gel showed presence of desired product
Members: Adam

September 12, 2012

Inoculated liquid cultures and plates with transformed E.Coli
Members: Adam

September 13, 2012

Performed ligation on Red Construct, Transformed E.Coli with blue construct
Members: Adam

September 14, 2012

Inoculated liquid cultures with blue construct transformants
Members: Adam

September 15, 2012

Ran mini-preps on colonies with blue constructs
Members: Adam and Bobby

September 16, 2012

Digested Blue construct and pRS413, isolated desired fragments from gel
Members: Adam

September 17, 2012

Ligated Blue construct and pRS413 fragments, transformed E.Coli
Members: Adam

September 18, 2012

Inoculated Liquid Media with Blue construct/pRS413 fusion bearing E.Coli
Members: Adam

September 19, 2012

NTNU construct testing via fluorescence, Mini-Preps of Blue construct/pRS413 fusion bearing E.Coli
Members: Bobby, Alex, Adam

September 20, 2012

Diagnostic gels of Blue Construct/pRS413 mini-preps
Members: Adam

September 22, 2012

Inoculated liquid media with Blue Construct/pRS413 E. Coli, prepared stock solutions
Members: Adam, Dr. A

September 23, 2012

Mini-Preps of E.Coli, Ran gel of prep, made liquid media
Members: Adam

September 24, 2012

Ran digestion of mini-prep DNA, plated Yeast
Members: Adam

September 25, 2012

Gel of digestion result, isolated fragments from gel
Members: Adam

September 26, 2012

Ligated Blue Fragment/pRS413, transformed E.Coli
Members: Adam

September 27, 2012

Inoculated LB-AMP broth with transformed E.Coli, plated Yeast
Members: Adam

September 28, 2012

Inoculated LB-CARB broth with transformed E.Coli
Members: Adam

September 29, 2012

Inoculated LB-CARB broth with transformed E.Coli
Members: Adam

September 30, 2012

Performed Mini-preps of transformed E.Coli, Began yeast transformation, plated additional yeast cultures.
Members: Adam

Strains Used

E. Coli

NEB5-alpha (NEB #C2988J or #C2987I): fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80Δ (lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17


S. Cerevisiae

BY4741 (ATCC #201388): MATa his3-Δ0 leu2-Δ0 met15-Δ0 ura3-Δ0


BY4742 (ATCC #201389): MATα his3-Δ0 leu2-Δ0 lys2-Δ0 ura3-Δ0


Transformations were carried out on competent E.Coli using the procedure available on the NEB website.


DNA extraction from cells was performed using a Qiagen QIAprep Spin Miniprep Kit (Cat. no. 27104). Included procedure was followed.


Digestions were performed using EcoRI, XbaI, SpeI and PstI using recommended NEB procedure.


Gel extraction of digested DNA was performed using a Qiagen QIAEX II Gel Extraction Kit (Cat. no. 20021). Included procedure was followed.


Ligations were performed following procedure available on the NEB website. Total mass of DNA for ligations was 100ng with a 1:3 to 1:4 ratio of vector to insert.


The official RHIT Safety page can be found here.

Researcher, Public, and Environmental Safety

The Rose-Hulman iGEM team has considered safety a top priority since the early stages of project development. As a result, projects that could pose a significant risk were dismissed. Our laboratory space is considered a Basic Biosafety Level 1 laboratory but it does have a few characteristics of higher categories. It has controlled access, biohazard signs and waste disposal bins, and an autoclave. All participating students received standard safety and good laboratory practice training as part of their academic laboratory course-work. The Instructor provided more specific safety instruction, as necessary. Proper attire was worn at all times in the laboratory. Gloves and glasses were donned as warranted. The only organisms used in our project are common laboratory strains of Saccharomyces cerevisiae (BY4741 and BY4742) and Escherichia coli (NEB5alpa), which are both considered Risk Group 1 microorganisms according to the Laboratory Biosafety Manual published by the World Health Organization. Group 1 organisms are defined as “no or low individual and community risk, a microorganism that is unlikely to cause human or animal disease.” Aseptic technique is used whenever working with these organisms and any contaminated wastes are sterilized by autoclaving or destroyed by commercial pyrolysis. All laboratory chemicals are stored, handled and used as recommended by the manufacturer, and they are disposed of in accordance with national, state, and local regulations and recommendations. The laboratory space and contents are not accessible by unauthorized personnel. All microbial strains, including bacteria rendered antibiotic resistant by transformation, harbor nutritional auxotrophies or other mutations that mitigate the risk of their growing outside of the laboratory or causing disease in healthy humans or animals. Furthermore, none of our recombinant constructs produce any known contagion or toxin.

BioBrick Safety

None of the BioBrick parts utilized or constructed are known to pose any safety issues. Furthermore, they are well contained by the microbes that harbor them. The risk of unintended transfer to any other organism is minimal.

Biosafety Group

Rose-Hulman does not have a biosafety group, committee or review board other than an Animal Care and Use Committee, which oversees animal research. Safety training and laboratory waste disposal are facilitated by an Environmental Health and Safety Officer, who also serves as a resource for faculty and students.

Future iGEM Safety

The possibility of designing mutually dependent strain/vector systems for routine manipulation and storage of BioBrick parts should be explored.

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