Team:RHIT/Notebook

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

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

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</ul>

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<li>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.</li><br />

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

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</ul>

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+

<li>6/18/2012: Optimized DNA sequences, found all of the filler DNA.</li><br />

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

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</ul>

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<li>6/25/2012: Group discussion, work on school website, organization of collected information.</li><br />

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

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<h4>Team Break Week</h4>

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

<li>7/10/2012: Worked on wiki.</li><br />

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</ul>

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<li>7/16/2012: Decided to use GeneArt for sequences instead of original DNA company.</li><br />

<li>7/18/2012: Brainstormed ideas for side projects while waiting for DNA shipment.</li><br />

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</ul>

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<li>7/23/2012: Devon and Kristen began planning the THCM exhibit.</li><br />

<li>7/24/2012: Continued work in Teams. Began intial research into pathway for modeling.</li><br />

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</ul>

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<li>7/30/2012: Ironed out wiki formatting, began adding content, continued Maya animation.</li><br />

<li>7/31/2012: Lab work, continued adding content to wiki.</li><br />

<li>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.</li><br />

<li>8/2/2012: Lab work, continued work in Teams.</li><br />

-

<li>8/3/2012: Lab work, continued work in Teams.</li~~><br /~~>

+

<li>8/3/2012: Lab work, continued work in Teams.</li>

</ul>

-

+

<li>8/6/2012: Canoe trip with IRC members, lab work.</li><br />

<li>8/7/2012: Lab work, focus on modeling information for NTNU, continued work in Teams.</li><br />

-

<li>8/8/2012: Second Skype meeting with NTNU~~, continued work in teams~~, conclusion and review of Maya animation, ~~lab~~ work.</li><br />

+

<li>8/8/2012: Lab work, Second Skype meeting with NTNU, conclusion and review of Maya animation.</li><br />

+

<li>8/9/2012: Lab work, continued work in Teams.</li><br />

+

<li>8/10/2012: Lab work, continued work in Teams.</li>

+

</ul>

+

+

+

<li>8/13/2012: Lab work, continued work in Teams.</li><br />

+

<li>8/14/2012: Lab work, continued work in Teams.</li><br />

+

<li>8/15/2012: Lab work, continued work in Teams.</li><br />

+

<li>8/16/2012: Lab work, plans made for continuing into the school year.</li><br />

+

<li>8/17/2012: Cleaned workspace, finalized plans for continuation.</li><br />

</ul>

</div></a>

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Ligated pRS413 and red construct, transformed E. Coli with ligated plasmid, inoculated liquid media with NTNU E. Coli<br />

Members: Bobby, Adam

-

+

<h3>August 15, 2012</h3>

+

Mini-Preps of NTNU construct, confirmed presence on gel, Digested pRS413, pRS416, and Red Construct, poured ampicillin plates<br />

+

Members: Bobby, Adam

+

<h3>August 16, 2012</h3>

+

Isolated pRS413, pRS416, and Red Construct from gel, began storing strains, DNA, and equipment<br />

+

Members: Bobby, Adam

+

<h3>August 17, 2012</h3>

+

Ran digestion of pRS413, pRS416, and Red Construct, stored remaining strains, DNA, and equipment<br />

+

Members: Bobby, Adam, Dr. A

+

<h3>August 28, 2012</h3>

+

Transformed E.Coli with BBa_E0422 reporter<br />

+

Members: Bobby, Adam, Dr. A

+

<h3>August 29, 2012</h3>

+

Digested and isolated Red Construct and pRS413 from gel, Inoculated liquid media with BBa_E0422 reporter<br />

+

Members: Adam

+

<h3>August 30, 2012</h3>

+

Digested pRS413 and Red Construct, Isolated from gel and Ligated, Transformed E. Coli.<br />

+

Members: Adam

+

<h3>August 31, 2012</h3>

+

Digested pRS413 and Red Construct<br />

+

Members: Bobby, Adam, Dr. A

+

<h3>September 1, 2012</h3>

+

Started cultures of pRS413 and the Red construct form frozen cultures<br />

+

Members: Adam

+

<h3>September 2, 2012</h3>

+

Inoculated liquid media with colonies from plates of pRS413 and Red Construct strains<br />

+

Members: Adam

+

<h3>September 3, 2012</h3>

+

Ran mini-preps of liquid colonies<br />

+

Members: Adam

+

<h3>September 4, 2012</h3>

+

Ran diagnostic gel to check amount of DNA extracted<br />

+

Members: Adam

+

<h3>September 5, 2012</h3>

+

Purified and concentrated DNA samples<br />

+

Members: Adam

+

<h3>September 6, 2012</h3>

+

Ran diagnostic gel to check for purification success<br />

+

Members: Adam

+

<h3>September 7, 2012</h3>

+

Ran BioBrick Assembly protocol on NTNU lld promoter and BBa_E0422, Transformed E.Coli with ligation mixture<br />

+

Members: Adam, Dr. A

+

<h3>September 8, 2012</h3>

+

Inoculated liquid cultures with transformed E.Coli<br />

+

Members: Adam

+

<h3>September 10, 2012</h3>

+

Digested and isolated Red Construct and pRS413 from gel, Ran mini-preps of liquid cultures<br />

+

Members: Adam

+

<h3>September 11, 2012</h3>

+

Ran gel of lld/0422 hybrid, Gel showed presence of desired product <br />

+

Members: Adam

+

<h3>September 12, 2012</h3>

+

Inoculated liquid cultures and plates with transformed E.Coli<br />

+

Members: Adam

+

<h3>September 13, 2012</h3>

+

Performed ligation on Red Construct, Transformed E.Coli with blue construct<br />

+

Members: Adam

+

<h3>September 14, 2012</h3>

+

Inoculated liquid cultures with blue construct transformants<br />

+

Members: Adam

+

<h3>September 15, 2012</h3>

+

Ran mini-preps on colonies with blue constructs<br />

+

Members: Adam and Bobby

+

<h3>September 16, 2012</h3>

+

Digested Blue construct and pRS413, isolated desired fragments from gel<br />

+

Members: Adam

+

<h3>September 17, 2012</h3>

+

Ligated Blue construct and pRS413 fragments, transformed E.Coli<br />

+

Members: Adam

+

<h3>September 18, 2012</h3>

+

Inoculated Liquid Media with Blue construct/pRS413 fusion bearing E.Coli<br />

+

Members: Adam

+

<h3>September 19, 2012</h3>

+

NTNU construct testing via fluorescence, Mini-Preps of Blue construct/pRS413 fusion bearing E.Coli<br />

+

Members: Bobby, Alex, Adam

+

<h3>September 20, 2012</h3>

+

Diagnostic gels of Blue Construct/pRS413 mini-preps<br />

+

Members: Adam

+

<h3>September 22, 2012</h3>

+

Inoculated liquid media with Blue Construct/pRS413 E. Coli, prepared stock solutions<br />

+

Members: Adam, Dr. A

+

<h3>September 23, 2012</h3>

+

Mini-Preps of E.Coli, Ran gel of prep, made liquid media<br />

+

Members: Adam

+

<h3>September 24, 2012</h3>

+

Ran digestion of mini-prep DNA, plated Yeast<br />

+

Members: Adam

+

<h3>September 25, 2012</h3>

+

Gel of digestion result, isolated fragments from gel<br />

+

Members: Adam

+

<h3>September 26, 2012</h3>

+

Ligated Blue Fragment/pRS413, transformed E.Coli<br />

+

Members: Adam

+

<h3>September 27, 2012</h3>

+

Inoculated LB-AMP broth with transformed E.Coli, plated Yeast<br />

+

Members: Adam

+

<h3>September 28, 2012</h3>

+

Inoculated LB-CARB broth with transformed E.Coli<br />

+

Members: Adam

+

<h3>September 29, 2012</h3>

+

Inoculated LB-CARB broth with transformed E.Coli<br />

+

Members: Adam

+

<h3>September 30, 2012</h3>

+

Performed Mini-preps of transformed E.Coli, Began yeast transformation, plated additional yeast cultures.<br />

+

Members: Adam

+

<h3>October 1, 2012</h3>

+

Plated Yeast Transformants<br />

+

Members: Adam

+

<h3>October 3, 2012</h3>

+

Transformed yeast found growing on plates<br />

+

Members: Adam

</div>

-

~~Protocol text~~

+

<h2>Strains Used</h2>

+

+

<p>NEB5-alpha (NEB #C2988J or #C2987I): <i>fhuA2 ?(argF-lacZ)U169 phoA glnV44 F80? (lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17</i></p><br />

+

<h3><i>S. Cerevisiae</i></h3>

+

+

<p>BY4742 (ATCC #201389): <i>MATalpha his3-?0 leu2-?0 lys2-?0 ura3-?0</i></p><br /><br />

+

<h2>Lab Protocols</h2>

+

<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 /><br />

+

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

+

<p>Digestions were performed using EcoRI, XbaI, SpeI and PstI using recommended NEB procedure.</p><br /><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><br /><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><br /><br />

+

</div>

+

The official RHIT Safety page can be found <a href="https://2012.igem.org/Team:RHIT/Safety">here</a>.

<h2>Researcher, Public, and Environmental Safety</h2>

-

<p>The Rose-Hulman iGEM team has ~~kept~~ safety a top priority since the ~~very~~ early stages of ~~the~~ project. As a result, ~~any project~~ that ~~might have posed as~~ a significant ~~threat to safety was quickly~~ dismissed. ~~During the planning phase~~ of ~~Checkmate~~, ~~team members brainstormed lab procedures as well as guidelines for plasmid construction to provide a safe~~ and ~~effective workplace. Despite best efforts though~~, ~~no project is without~~ safety ~~concerns~~. ~~Some of the lab methods require using potentially hazardous chemicals that could cause injury to any one of the researchers~~. ~~Another potential safety risk for researchers results from dealing with microbial agents~~. ~~While it is unlikely that a researcher would develop an infection from the strains he or she is working with, contagion is still a possible~~ safety ~~concern~~. ~~To combat these risks, the team was sure to follow all~~ safety ~~protocols~~, ~~from aseptic techniques to complete labeling~~. Proper ~~lab~~ attire was worn at all times in the ~~lab~~.

+

<p>The Rose-Hulman iGEM team has considered safety a top priority since the early stages of project development.

-

~~When considering public safety, it is important to consider every aspect and intermediate step of the~~ project~~. In Checkmate, the~~ strains of ~~yeast used contain a knockout for an essential amino acid. As a result of this knockout, it is highly unlikely that this yeast could ever escape the lab~~ and ~~be released into the public. However~~, ~~not all the species involved in the project~~ are ~~so easily controlled. The selection scheme for maintaining~~ the ~~plasmid in E. Coli involved providing~~ the ~~bacteria with antibiotic resistance~~. ~~In an age where multiple drug resistant bacteria~~ are ~~on the rise~~ and ~~responsible for killing several thousand people a year~~, ~~the potential of this bacteria escaping and passing this plasmid on~~ to ~~another bacteria cannot be ignored. When handling E~~. ~~Coli, the team~~ used ~~aseptic techniques~~ and sterilized ~~their equipment~~.

+

As a result, projects that could pose a significant risk were dismissed.

-

~~Similar to~~ the ~~public safety concerns~~, ~~most~~ of ~~the environmental safety concerns stem from chemicals or biological agents leaving the lab~~ and ~~being exposed to the environment outside the lab~~. ~~Improper disposal~~ or ~~contamination of~~ the ~~environment by a couple~~ of ~~Checkmate’s chemicals could prove deleterious to~~ outside ~~environments~~. ~~Once again~~, ~~sterilization and aseptic techniques prevented~~ any ~~calamity~~.</p>

+

Our laboratory space is considered a Basic Biosafety Level 1 laboratory but it does have a few characteristics of higher categories.

-

<h2>BioBrick Safety</h2>

+

It has controlled access, biohazard signs and waste disposal bins, and an autoclave. Other safety features are demonstrated below.<br /></p>

-

<p>~~To ensure the safety~~ of ~~our BioBricks, we consulted~~ the BioBrick ~~standard for regulation of restriction sites when designed the DNA sequence~~. ~~In addition~~, *SENTENCE TALKING ABOUT VECTOR KNOCKOUT OF KEY AMINO ACID. ~~By characterizing the BioBricks~~ to ~~their fullest potential, it will allow~~ other ~~participates of iGEM to use our BioBricks in their intended way, optimizing the efficiency of their projects~~.</p>

+

-

<h2>Biosafety Group</h2>

+

<p>All participating students received standard safety and good laboratory practice training as part of their academic laboratory course-work.

-

<p>Rose-Hulman ~~Institute of Technology, due to a small applied biology program,~~ does not have a biosafety group. ~~Instead, safety procedures~~ are ~~integrated into classes~~ by ~~professors. The United States of America has many biosafety rules about working with human cells~~, ~~but projects that work without human cells, such~~ as ~~Checkmate, are not regulated by these rules because they do not pose the same threat as projects that use human cells~~.</p>

+

The Instructor provided more specific safety instruction, as necessary.

-

<h2>Future Safety</h2>

+

Proper attire was worn at all times in the laboratory. Gloves and glasses were donned as warranted.

-

<p>~~Future iGEM teams can be safe by following appropriate lab safety procedures. When considering their project, future iGEM teams need to be aware that they are creating something new,~~ and should ~~proceed with caution, always keeping safety at the forefront. Specifically, one way to~~ be sure that iGEM projects cannot be a danger to the public or the environment is to include some form of self-destruct. This would make the cells unable to live in a non-lab environment. The DNA could be degenerative over time, or a strain could be dependent on a vital nutrient only found in a laboratory setting.</p>

+

The only organisms used in our project are common laboratory strains of <i>Saccharomyces cerevisiae</i> (BY4741 and BY4742) and <i>Escherichia coli</i> (NEB5alpa),

-

</div>

+

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.<p>

+

<h2>BioBrick Safety</h2>

+

<p>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.</p>

+

<h2>Biosafety Group</h2>

+

<p>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.</p>

+

<h2>Future iGEM Safety</h2>

+

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

+

</div>

</body>

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~~</html>~~

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+							$('#rhit-sheet2').hide();
+var split = location.search.replace('?', '').split('=');
+window[split[1]]();
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+			function page1() {
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 		<div class="rhit-content">
 			<div class="rhit-header">
-				<img src="https://static.igem.org/mediawiki/igem.org/8/87/Banner_only2.png" width="100%"/>
+				<a href="https://2012.igem.org/Team:RHIT"><img src="https://static.igem.org/mediawiki/igem.org/8/87/Banner_only2.png" width="100%"/></a>
 				<div id="logged_out">
 					<a href="https://2012.igem.org/Main_Page">iGEM Home</a>
@@ Line 102: / Line 105: @@
 				<div class="rhit-blueSheet" id="rhit-blueSheet">
 					<h4>Week 1</h4>
-					<ul id="dailyLog" style="list-style-type:none">
+					<ul id="dailyLog">
 					<li>6/4/2012: First round of project planning, lab clean-up, general outline for summer.</li><br />
 					<li>6/5/2012: Second round of project planning, lab clean-up, research into Johns Hopkins 2008 summer project.</li><br />
@@ Line 110: / Line 113: @@
 					</ul>
 					<h4>Week 2</h4>
-					<ul id="dailyLog" style="list-style-type:none">
+					<ul id="dailyLog">
 					<li>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.</li><br />
 					<li>6/12/2012: Found gene sequences, continued work on graphic for top of Wiki, looked for selection schemes, shot down selection schemes.</li><br />
@@ Line 118: / Line 121: @@
 					</ul>
 					<h4>Week 3</h4>
-					<ul id="dailyLog" style="list-style-type:none">
+					<ul id="dailyLog">
 					<li>6/18/2012: Optimized DNA sequences, found all of the filler DNA.</li><br />
 					<li>6/19/2012: Talked with Dr. Almaas, started web design, took pictures with storm troopers, team movie night.</li><br />
@@ Line 126: / Line 129: @@
 					</ul>
 					<h4>Week 4</h4>
-					<ul id="dailyLog" style="list-style-type:none">
+					<ul id="dailyLog">
 					<li>6/25/2012: Group discussion, work on school website, organization of collected information.</li><br />
 					<li>6/26/2012: Obtained shirts, found and organized all sequences, started to verify sequences, school page is 85% done.</li><br />
@@ Line 135: / Line 138: @@
 					<h4>Team Break Week</h4>
 					<h4>Week 5</h4>
-					<ul id="dailyLog" style="list-style-type:none">
+					<ul id="dailyLog">
 					<li>7/9/2012: Got back from break. Learned DNA had not shipped yet. :( Worked on wiki.</li><br />
 					<li>7/10/2012: Worked on wiki.</li><br />
@@ Line 143: / Line 146: @@
 					</ul>
 					<h4>Week 6</h4>
-					<ul id="dailyLog" style="list-style-type:none">
+					<ul id="dailyLog">
 					<li>7/16/2012: Decided to use GeneArt for sequences instead of original DNA company.</li><br />
 					<li>7/18/2012: Brainstormed ideas for side projects while waiting for DNA shipment.</li><br />
@@ Line 151: / Line 154: @@
 					</ul>
 					<h4>Week 7</h4>
-					<ul id="dailyLog" style="list-style-type:none">
+					<ul id="dailyLog">
 					<li>7/23/2012: Devon and Kristen began planning the THCM exhibit.</li><br />
 					<li>7/24/2012: Continued work in Teams. Began intial research into pathway for modeling.</li><br />
@@ Line 159: / Line 162: @@
 					</ul>
 					<h4>Week 8</h4>
-					<ul id="dailyLog" style="list-style-type:none">
+					<ul id="dailyLog">
 					<li>7/30/2012: Ironed out wiki formatting, began adding content, continued Maya animation.</li><br />
 					<li>7/31/2012: Lab work, continued adding content to wiki.</li><br />
 					<li>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.</li><br />
 					<li>8/2/2012: Lab work, continued work in Teams.</li><br />
-					<li>8/3/2012: Lab work, continued work in Teams.</li><br />
+					<li>8/3/2012: Lab work, continued work in Teams.</li>
 					</ul>
 					<h4>Week 9</h4>
-					<ul id="dailyLog" style="list-style-type:none">
+					<ul id="dailyLog">
 					<li>8/6/2012: Canoe trip with IRC members, lab work.</li><br />
 					<li>8/7/2012: Lab work, focus on modeling information for NTNU, continued work in Teams.</li><br />
-					<li>8/8/2012: Second Skype meeting with NTNU, continued work in teams, conclusion and review of Maya animation, lab work.</li><br />
+					<li>8/8/2012: Lab work, Second Skype meeting with NTNU, conclusion and review of Maya animation.</li><br />
+					<li>8/9/2012: Lab work, continued work in Teams.</li><br />
+					<li>8/10/2012: Lab work, continued work in Teams.</li>
+					</ul>
+					<h4>Week 10</h4>
+					<ul id="dailyLog">
+					<li>8/13/2012: Lab work, continued work in Teams.</li><br />
+					<li>8/14/2012: Lab work, continued work in Teams.</li><br />
+					<li>8/15/2012: Lab work, continued work in Teams.</li><br />
+					<li>8/16/2012: Lab work, plans made for continuing into the school year.</li><br />
+					<li>8/17/2012: Cleaned workspace, finalized plans for continuation.</li><br />
 					</ul>
 				</div></a>
@@ Line 213: / Line 226: @@
 					Ligated pRS413 and red construct, transformed E. Coli with ligated plasmid, inoculated liquid media with NTNU E. Coli<br />
 					Members: Bobby, Adam
+					<h3>August 15, 2012</h3>
+					Mini-Preps of NTNU construct, confirmed presence on gel, Digested pRS413, pRS416, and Red Construct, poured ampicillin plates<br />
+					Members: Bobby, Adam
+					<h3>August 16, 2012</h3>
+					Isolated pRS413, pRS416, and Red Construct from gel, began storing strains, DNA, and equipment<br />
+					Members: Bobby, Adam
+					<h3>August 17, 2012</h3>
+					Ran digestion of pRS413, pRS416, and Red Construct, stored remaining strains, DNA, and equipment<br />
+					Members: Bobby, Adam, Dr. A
+					<h3>August 28, 2012</h3>
+					Transformed E.Coli with BBa_E0422 reporter<br />
+					Members: Bobby, Adam, Dr. A
+					<h3>August 29, 2012</h3>
+					Digested and isolated Red Construct and pRS413 from gel, Inoculated liquid media with BBa_E0422 reporter<br />
+					Members: Adam
+					<h3>August 30, 2012</h3>
+					Digested pRS413 and Red Construct, Isolated from gel and Ligated, Transformed E. Coli.<br />
+					Members: Adam
+					<h3>August 31, 2012</h3>
+					Digested pRS413 and Red Construct<br />
+					Members: Bobby, Adam, Dr. A
+					<h3>September 1, 2012</h3>
+					Started cultures of pRS413 and the Red construct form frozen cultures<br />
+					Members: Adam
+					<h3>September 2, 2012</h3>
+					Inoculated liquid media with colonies from plates of pRS413 and Red Construct strains<br />
+					Members: Adam
+					<h3>September 3, 2012</h3>
+					Ran mini-preps of liquid colonies<br />
+					Members: Adam
+					<h3>September 4, 2012</h3>
+					Ran diagnostic gel to check amount of DNA extracted<br />
+					Members: Adam
+					<h3>September 5, 2012</h3>
+					Purified and concentrated DNA samples<br />
+					Members: Adam
+					<h3>September 6, 2012</h3>
+					Ran diagnostic gel to check for purification success<br />
+					Members: Adam
+					<h3>September 7, 2012</h3>
+					Ran BioBrick Assembly protocol on NTNU lld promoter and BBa_E0422, Transformed E.Coli with ligation mixture<br />
+					Members: Adam, Dr. A
+					<h3>September 8, 2012</h3>
+					Inoculated liquid cultures with transformed E.Coli<br />
+					Members: Adam
+					<h3>September 10, 2012</h3>
+					Digested and isolated Red Construct and pRS413 from gel, Ran mini-preps of liquid cultures<br />
+					Members: Adam
+					<h3>September 11, 2012</h3>
+					Ran gel of lld/0422 hybrid, Gel showed presence of desired product <br />
+					Members: Adam
+					<h3>September 12, 2012</h3>
+					Inoculated liquid cultures and plates with transformed E.Coli<br />
+					Members: Adam
+					<h3>September 13, 2012</h3>
+					Performed ligation on Red Construct, Transformed E.Coli with blue construct<br />
+					Members: Adam
+					<h3>September 14, 2012</h3>
+					Inoculated liquid cultures with blue construct transformants<br />
+					Members: Adam
+					<h3>September 15, 2012</h3>
+					Ran mini-preps on colonies with blue constructs<br />
+					Members: Adam and Bobby
+					<h3>September 16, 2012</h3>
+					Digested Blue construct and pRS413, isolated desired fragments from gel<br />
+					Members: Adam
+					<h3>September 17, 2012</h3>
+					Ligated Blue construct and pRS413 fragments, transformed E.Coli<br />
+					Members: Adam
+					<h3>September 18, 2012</h3>
+					Inoculated Liquid Media with Blue construct/pRS413 fusion bearing E.Coli<br />
+					Members: Adam
+					<h3>September 19, 2012</h3>
+					NTNU construct testing via fluorescence, Mini-Preps of Blue construct/pRS413 fusion bearing E.Coli<br />
+					Members: Bobby, Alex, Adam
+					<h3>September 20, 2012</h3>
+					Diagnostic gels of Blue Construct/pRS413 mini-preps<br />
+					Members: Adam
+					<h3>September 22, 2012</h3>
+					Inoculated liquid media with Blue Construct/pRS413 E. Coli, prepared stock solutions<br />
+					Members: Adam, Dr. A
+					<h3>September 23, 2012</h3>
+					Mini-Preps of E.Coli, Ran gel of prep, made liquid media<br />
+					Members: Adam
+					<h3>September 24, 2012</h3>
+					Ran digestion of mini-prep DNA, plated Yeast<br />
+					Members: Adam
+					<h3>September 25, 2012</h3>
+					Gel of digestion result, isolated fragments from gel<br />
+					Members: Adam
+					<h3>September 26, 2012</h3>
+					Ligated Blue Fragment/pRS413, transformed E.Coli<br />
+					Members: Adam
+					<h3>September 27, 2012</h3>
+					Inoculated LB-AMP broth with transformed E.Coli, plated Yeast<br />
+					Members: Adam
+					<h3>September 28, 2012</h3>
+					Inoculated LB-CARB broth with transformed E.Coli<br />
+					Members: Adam
+					<h3>September 29, 2012</h3>
+					Inoculated LB-CARB broth with transformed E.Coli<br />
+					Members: Adam
+					<h3>September 30, 2012</h3>
+					Performed Mini-preps of transformed E.Coli, Began yeast transformation, plated additional yeast cultures.<br />
+					Members: Adam
+					<h3>October 1, 2012</h3>
+					Plated Yeast Transformants<br />
+					Members: Adam
+					<h3>October 3, 2012</h3>
+					Transformed yeast found growing on plates<br />
+					Members: Adam
 				</div>
 				<div class="rhit-grnSheet" id="rhit-grnSheet">
-					Protocol text
+					<h2>Strains Used</h2>
+					<h3><i>E. Coli</i></h3>
+					<p>NEB5-alpha (NEB #C2988J or #C2987I): <i>fhuA2 ?(argF-lacZ)U169 phoA glnV44 F80? (lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17</i></p><br />
+					<h3><i>S. Cerevisiae</i></h3>
+					<p>BY4741 (ATCC #201388): <i>MATa his3-?0 leu2-?0 met15-?0 ura3-?0</i></p><br />
+					<p>BY4742 (ATCC #201389): <i>MATalpha his3-?0 leu2-?0 lys2-?0 ura3-?0</i></p><br /><br />
+					<h2>Lab Protocols</h2>
+					<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 /><br />
+					<p>DNA extraction from cells was performed using a Qiagen QIAprep Spin Miniprep Kit (Cat. no. 27104). Included procedure was followed.</p><br /><br />
+					<p>Digestions were performed using EcoRI, XbaI, SpeI and PstI using recommended NEB procedure.</p><br /><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><br /><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><br /><br />
 				</div>
 				<div class="rhit-fourthSheet" id="rhit-fourthSheet">
+					The official RHIT Safety page can be found <a href="https://2012.igem.org/Team:RHIT/Safety">here</a>.
 					<h2>Researcher, Public, and Environmental Safety</h2>
-<p>The Rose-Hulman iGEM team has kept safety a top priority since the very early stages of the project. As a result, any project that might have posed as a significant threat to safety was quickly dismissed. During the planning phase of Checkmate, team members brainstormed lab procedures as well as guidelines for plasmid construction to provide a safe and effective workplace. Despite best efforts though, no project is without safety concerns. Some of the lab methods require using potentially hazardous chemicals that could cause injury to any one of the researchers. Another potential safety risk for researchers results from dealing with microbial agents. While it is unlikely that a researcher would develop an infection from the strains he or she is working with, contagion is still a possible safety concern. To combat these risks, the team was sure to follow all safety protocols, from aseptic techniques to complete labeling. Proper lab attire was worn at all times in the lab.
+					<p>The Rose-Hulman iGEM team has considered safety a top priority since the early stages of project development.
-When considering public safety, it is important to consider every aspect and intermediate step of the project. In Checkmate, the strains of yeast used contain a knockout for an essential amino acid. As a result of this knockout, it is highly unlikely that this yeast could ever escape the lab and be released into the public. However, not all the species involved in the project are so easily controlled. The selection scheme for maintaining the plasmid in E. Coli involved providing the bacteria with antibiotic resistance. In an age where multiple drug resistant bacteria are on the rise and responsible for killing several thousand people a year, the potential of this bacteria escaping and passing this plasmid on to another bacteria cannot be ignored. When handling E. Coli, the team used aseptic techniques and sterilized their equipment.
+					As a result, projects that could pose a significant risk were dismissed.
-Similar to the public safety concerns, most of the environmental safety concerns stem from chemicals or biological agents leaving the lab and being exposed to the environment outside the lab. Improper disposal or contamination of the environment by a couple of Checkmate’s chemicals could prove deleterious to outside environments. Once again, sterilization and aseptic techniques prevented any calamity.</p>
+					Our laboratory space is considered a Basic Biosafety Level 1 laboratory but it does have a few characteristics of higher categories.
-<h2>BioBrick Safety</h2>
+					It has controlled access, biohazard signs and waste disposal bins, and an autoclave. Other safety features are demonstrated below.<br /></p>
-<p>To ensure the safety of our BioBricks, we consulted the BioBrick standard for regulation of restriction sites when designed the DNA sequence. In addition, *SENTENCE TALKING ABOUT VECTOR KNOCKOUT OF KEY AMINO ACID.  By characterizing the BioBricks to their fullest potential, it will allow other participates of iGEM to use our BioBricks in their intended way, optimizing the efficiency of their projects.</p>
+					<div align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/2a/Safety_pics.png" width="60%"></div><br />
-<h2>Biosafety Group</h2>
+					<p>All participating students received standard safety and good laboratory practice training as part of their academic laboratory course-work.
-<p>Rose-Hulman Institute of Technology, due to a small applied biology program, does not have a biosafety group. Instead, safety procedures are integrated into classes by professors. The United States of America has many biosafety rules about working with human cells, but projects that work without human cells, such as Checkmate, are not regulated by these rules because they do not pose the same threat as projects that use human cells.</p>
+					The Instructor provided more specific safety instruction, as necessary.
-<h2>Future Safety</h2>
+					Proper attire was worn at all times in the laboratory. Gloves and glasses were donned as warranted.
-<p>Future iGEM teams can be safe by following appropriate lab safety procedures. When considering their project, future iGEM teams need to be aware that they are creating something new, and should proceed with caution, always keeping safety at the forefront. Specifically, one way to be sure that iGEM projects cannot be a danger to the public or the environment is to include some form of self-destruct. This would make the cells unable to live in a non-lab environment. The DNA could be degenerative over time, or a strain could be dependent on a vital nutrient only found in a laboratory setting.</p>
+					The only organisms used in our project are common laboratory strains of <i>Saccharomyces cerevisiae</i> (BY4741 and BY4742) and <i>Escherichia coli</i> (NEB5alpa),
-				</div>
+					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.<p>
+					<h2>BioBrick Safety</h2>
+					<p>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.</p>
+					<h2>Biosafety Group</h2>
+					<p>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.</p>
+					<h2>Future iGEM Safety</h2>
+					<p>The possibility of designing mutually dependent strain/vector systems for routine manipulation and storage of BioBrick parts should be explored.</p>
+				</div>
 			</div>
 		</div>
 	</body>
-</html>

From 2012.igem.org

Latest revision as of 03:57, 4 October 2012

Week 1

Week 2

Week 3

Week 4

Team Break Week

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

July 31, 2012

August 1, 2012

August 2, 2012

August 3, 2012

August 6, 2012

August 7, 2012

August 8, 2012

August 9, 2012

August 10, 2012

August 11, 2012

August 12, 2012

August 13, 2012

August 14, 2012

August 15, 2012

August 16, 2012

August 17, 2012

August 28, 2012

August 29, 2012

August 30, 2012

August 31, 2012

September 1, 2012

September 2, 2012

September 3, 2012

September 4, 2012

September 5, 2012

September 6, 2012

September 7, 2012

September 8, 2012

September 10, 2012

September 11, 2012

September 12, 2012

September 13, 2012

September 14, 2012

September 15, 2012

September 16, 2012

September 17, 2012

September 18, 2012

September 19, 2012

September 20, 2012

September 22, 2012

September 23, 2012

September 24, 2012

September 25, 2012

September 26, 2012

September 27, 2012

September 28, 2012

September 29, 2012

September 30, 2012

October 1, 2012

October 3, 2012

Strains Used

E. Coli

S. Cerevisiae

Lab Protocols

Researcher, Public, and Environmental Safety

BioBrick Safety

Biosafety Group

Future iGEM Safety