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Team:TacomaRAINmakers/Notebook - 2017.igem.org

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Team:RAINmakers/Notebook

Tacoma RAINmakers Lab Notebook

Week 1

Digestion and Isolation of pSB1C3 Backbone.

The purpose of digesting the pSB1C3/PArsRGFP construct was to separate the backbone from the former GFP insert. Tacoma RAINmakers sought to isolate the pSB1C3 backbone employed in their 2017 construct, as the GFP reporter complex was no longer desired. Apparent disadvantages of GFP indication in biosensors are ultraviolet readings. The RAINmakers prefered a chromoprotein that produces color in the visible spectrum. Enzymes XbaI and SpeI were used to cleave the terminator sites of the vector, freeing the pSB1C3 backbone. NEB resources confirmed that the Cutsmart Buffer 2.1 is the most compatible with these particular enzymes, providing an optimized environment for digestion. Combining the reagents listed in Table 1.0, the reaction was set at 37ºC in a water bath for 1 hour and 45 minutes. This reaction was completed in duplicate to increase statistical probability of desired backbone DNA.

Following completion of pSB1C3 digestion, Tacoma RAINmakers employed a standard procedure listed in the protocol page as “Agarose Gel Electrophoresis.” The purpose of this gel was to confirm if the backbone DNA had been successfully isolated from the undesired GFP insert. As cited in Figure 1, the expected pSB1C3 bands were located at 2000bp. A gel extraction process, also outlined in the protocol section, was completed in order to remove and contain the digested pSB1C3 DNA.

Fig. 1. DNA gel of digested parts.

Week 2

Digestion and Ligation of spisPink, amilCP, and PcArsR Inserts

Using IDT stock solutions of chromoprotein and arsenic regulatory DNA, Tacoma RAINmakers completed a standard restriction digest (see protocol page for further information). The notable difference between insert and backbone digestion are the enzymes. Each insert included a SalI enzyme site, which is not compatible with the BioBrick suffix/prefix of the vector. Instead, the SalI site is used to ligate the chromoprotein to PcArsR, rendering the ends of this complete insert as compatible for sticky-end ligation to pSB1C3. After combining reagents listed in Table 2.0, reaction was held in a water bath at 37°C overnight.

Once the SalI site in spisPink, amilCP, and PcArsR was successfully sticky-ended, Tacoma RAINmakers were prepared for ligation of each chromoprotein to the arsenic regulator. 60ng of each part were used in order to ensure that there would enough DNA material for the ligation. Having combined the substances from Table 2.1, the reaction was set to ligate overnight at 16°C. Afterwards, SalI was heat inactivated at 80°C, ensuring a complete denature, since the enzyme was no longer required.

Week 3

Digestion and Ligation of Insert (amilCP/spisPink + PcArsR) to pSB1C3 Backbone

Following the ligation of the chromoprotein and PcArsR, the complete insert was digested with enzymes compatible with our pSB1C3 backbone. This process allows sticky-ended ligation in the next step, which increases the chance of a proper insert-backbone ligation. 84 ng of insert DNA was pipetted into the reaction alongside the other reagents mentioned in Table 3.0. The digestion was set at 37°C in a water bath for 1 hour and 30 minutes.

Tacoma RAINmakers combined the reagents listed in Table 3.1 to ligate the completed insert to the vector. The reaction included a negative control that contained only vector DNA. A notable process involved in ligation reactions is calculating DNA volumes. Typically, a 1:3 ratio of vector to insert ensures that there is a balance of both parts. The RAINmakers employed the NEBioCalculator to determine how many moles were in 1ng of vector (2070bp) and 1ng of insert (1488bp). This calculation translated to 0.8µL of vector and 20µL of insert. The ligation occurred at 16ºC overnight and was heat inactivated at 80ºC for 20 minutes the following morning.

Initiation of Individual Chromoprotein and Regulator Plasmid Design

A standard procedure in the Tacoma RAINmakers project is PCR amplification. This process is listed under the protocol page as “Insert PCR Amplification.” In preparation for ligation of inserts (amilCP, spisPink, and PcArsR) into the vector, all insert DNA must be amplified from its original limited stock. Once the PCR reaction has exited the thermocycler, gel electrophoresis must be employed to assess the efficacy of the amplification. As pictured in Figure 3, both the spisPink and amilCP bands successfully appeared at about 1000bp, and the PcArsR expressed at about 550bp. Unfortunately, the PcArsR negative control produced DNA bands, which suggested contamination during the PCR amplification process.

Following a successful PCR amplification confirmed by the gel, Tacoma RAINmakers performed a standard gel extraction. With much more insert DNA, RAINmakers were prepared to design three additional plasmids containing single inserts for isolated testing.

Fig. 2. DNA gel of PCR products.

Week 4

Transformation of Arsenic Construct and PCR Screening

TThe positive control (pSB1C3 + Insert) and negative control (pSB1C3 only) were transformed using DH5 Alpha Competent E. coli cells. Although the iGEM protocol for transformation states that 1µL of DNA is sufficient, 2µL of DNA were used for both the positive and negative control plates to ensure enough DNA existed for multiple colonies to grow. Transformation reactions were incubated overnight at 37ºC within a 14-18 hour time period. An extended description of the standard transformation process is listed under the protocol page.
The RAINmakers ran a PCR screening for both the positive (vector and insert) and negative (vector only) controls. This PCR allows us to amplify our cloned DNA, which is necessary to do because it will help us determine whether or not the ligation of the vector and insert was successful. The product after our PCR will be used to run a gel, which will help us see which colonies have the vector and insert and which only have the vector. After determining the volumes of the reagents, we decided to add an extra 20% uncertainty to each reagent in the master mix since small volumes of liquid can get stuck in the pipette. We did all these calculations based on the fact that we planned on doing 8 PCR reactions (8 PCR tubes). Before beginning the process of adding all the reagents to our PCR tubes, we needed to determine how long the extension part of PCR should be based on the base pair count of our product. Running a PCR simulation of Snapgene allowed us to find the exact base pair count of our positive control, which is 1597 base pairs. From this, we found that the extension period should be 1 minute and 36 seconds.
After adding all of our reagents together into 8 PCR tubes, we needed to select single colonies from transformation plates from 6/19/18. We determined that 6 of our PCR tubes would have the positive control, 1 would be the negative control and the last tube would have no DNA. Once we selected a single colony and put the DNA into a PCR tube, we streaked it onto a new plate (cut up in 8 different sections for each 8 different colonies) and labeled them 1, 2, 3, 4, 5, 6 (positive control), 7 (nothing) and 8 (negative control). We then incubated this new plate so that more colonies would grow. Finally, we put our PCR tubes in a thermocycler and ran our PCR.

Blunt-End Digestion and Ligation of pSB1C3 and Inserts

Designing plasmids with individual inserts becomes slightly complicated, as each insert contains a SalI site that is not compatible with the BioBrick prefix/suffix of the backbone. Hence, Tacoma RAINmakers preferred blunt-end ligation, rendering the incompatible ends irrelevant.
Additionally, each insert was digested with its respective enzyme and filled in with T4 polymerase in preparation for blunt-end ligation.

Week 5 - Week 7

We re-started the cloning process with fresh PCR products and more vector. We re-ligated our pSB1C3 backbone with our PcArsR+SpisPink/PcArsR+amilCP insert several times with only negative results. We decided to re-ligate PcArsR + chromoprotein (spisPink or amilCP), as there were speculations that something went wrong in the initial ligation of the two. This ligation contained 3 uL of PcArsR, 3 uL of chromoprotein (spisPink or amilCP), 1x T4 DNA Ligase Buffer, 1.5U T4 DNA Ligase, and molecular water to a final volume of 10 uL. We ligated at 16ºC overnight, then heat killed at 80ºC for 20 minutes.

After this ligation was complete, we digested the newly ligated insert with XbaI and SpeI to get it ready for ligation with the pSB1C3 backbone. This digestion contained 1 uL of Buffer 2.1, 1 uL of XbaI, 1 uL of SpeI, and the newly ligated insert DNA. We did an overnight transformation and then ran PCR on the colonies. To see the results we ran it on a 1% agarose gel and saw that the insert did not ligate to the vector. This sequence of events repeated itself for many weeks, to great frustration.

Week 8

Restart cloning process

After failed attempts to clone the circuit, we decided to start again from the beginning. In the digestion of the backbone, we followed the iGEM protocol, but modified it by doing an overnight digestion at 16ºC instead of a 30 minute digestion. The same digestion was performed on PcArsR, spisPink, and AmilCP. We heat killed the ligase at 80ºC for 20 minutes the next day, and digested the new insert with XbaI and SpeI. After digestion, we did an overnight ligation of the vector and the insert using the slightly modified iGEM protocol with an overnight incubation.

Testing ligation success before transformation

The team spent a lot of time transforming without successful insertion of the circuit, and needed a better way to determine the quality of ligation product that was being put into the cells. We reasoned that since PCR can amplify the tiniest amounts of DNA, we may be able to use this to screen the ligation products.
To screen the ligation, we ran end point PCR on the newly ligated parts using our standard PCR protocol. Surprisingly, the PCR confirmed that the vector and insert successfully ligated together! We then transformed our full circuit using the transformation protocol on the iGEM website, and mini-prepped using the Promega Wizard Plus SV Kit.

Fig. 3. DNA gel of ligation showing successful ligation.

Week 9

Bioengineering Summer Camp

The team took a week off from lab work to run a summer bioengineering camp for 9th and 10th grade students. We had a lot of fun teaching the students and used many tools gained from iGEM, like letting students streak out bacteria containing RFP (positive transformation control) and GFP (interlab positive control).

Week 10

Circuit testing

We started the process to test the complete arsenic circuit. In vivo testing of full circuits with reporters amilCP and spisPink was performed with 1uM/2uM/10uM concentrations of arsenate/arsenite. RAIN's 2017 iGEM team had ran an experiment testing 150uM arsenic which killed the cells during the experiment. A second experiment was conducted where 25uM arsenic was used. This did not kill the cells and GFP was produced. This is the maximum amount of arsenic we want to test.

The limits of detection for our circuit to be viable and to compete with current testing equipment is below 100ppb. At 50ppb soil and water is considered unsafe for consumption or play. At 100ppb the soil or water is deemed worthy for excavation due to its potential health risks. Therefore, we will test 1uM arsenic (equivalent to 75ppb), 2uM arsenic (150ppb) and 10uM arsenic (750ppb).

10mL of liquid culture was created for both spisPink and amilCP the cultures will be set up as follows:

The cultures were observed at 1 hour, 3 hours, 24 hours and 48 hours for visual solution color change. During the entirety of the experiment, no color change or production occurred.

Repeat circuit testing with higher concentration of arsenite/arsenate

Testing of the amilCP and spisPink in vivo was unsuccessful at 1uM/2uM/10uM. There was also an attempt to grow CFU plates, however, these plates were left in the incubator for two days and overgrew. RAIN's 2017 iGEM team was able to see production of GFP at 25uM concentration of arsenic so this experiment will lie closer to these values.

Three concentrations or arsenic will be tested this time: 17.65uM (equivalent to 1324ppb), 21.42uM (1606ppb) and 25uM (1875ppb).
4mL of liquid culture was created for both spisPink and amilCP circuits.

After growing overnight, there were no color changes in the cultures.

Week 11

More cloning

Since we had a lot of trouble seeing any reporter expression, the team wanted to clone the individual parts of just the regulator and just the reporters. This will allow us to see if the reporter will turn on when its not being repressed with ArsR. Unfortunately, we ran into the same problems as before. By this, we mean that the ligation reactions were not demonstrating successful insertion of our DNA into the vector.

Week 12-13

Different members of the team kept trying to clone the individual parts. The individual parts were amplified using our standard PCR protocol. Following PCR amplification, the reactions were run at 120V on a 1% Agarose gel. After seeing in the light box that the PCR was successful, the PCR products were extracted using a standard protocol and stored at -20°C.

Fig. 4. DNA gel of PCR products.

The purified parts and pSB1C3 were digested overnight with XbaI and SpeI. The ligation was performed the next day using reactions contained 4.1μL of digested insert, 1.35μL digested linear pSB1C3, 1u T4 DNA ligase, and 1x T4 ligase buffer, and Molecular Water to a final volume of 10μL. It was incubated overnight at 16C. Then, the ligation was tested with PCR primers amplifying the region between the XbaI and SpeI sites on pSB1C3. The PCR reactions contained 1x GoTaq Flexi Green Buffer, 0.15mM DNTP, 1.5mM MgCl2, 0.63u GoTaq Polymerase, 0.5μL of Ligation Product, 0.38μM FWD primer, 0.38μM REV primer, and molecular water to a final volume of 10μL. We ran it on a 1% agarose gel and saw no bands of the desired size.

In order to test the amilCP reporter, we created a circuit with a high expression T7 promoter, amilCP, and the same double terminator used in our other circuits. We began cloning by digesting IDT stock solutions of T7/amilCP with EcoRI-HF and PstI, following iGEM standard protocols. We then ligated T7/amilCP with pSB1C3, using the iGEM ligation protocol. To screen the ligation we used our PCR protocol. After running PCR products on an agarose gel, we found that the ligation failed.

Week 14

We repeated the ligation at 4°C overnight. The reactions contained 5μL digested insert, 5μL digested linear pSB1C3, 1U T4 DNA Ligase, 1x T4 Ligase Buffer, and molecular water to a final volume of 15μL. The ligation was tested in the same way, and once again produced no bands.

The third attempt at ligation contained 10μL digested insert, 2.5μL of a plasmid with pSB1C3 that we digested using XbaI and SpeI, 1u T4 DNA Ligase, 1x T4 Ligase Buffer, and molecular water to a final volume of 20μL. It was left at room temperature for 1 hour, then moved to incubate at 16°C overnight. The ligation was tested in the same way as the previous 2 attempts. The gel had strong bands at 75bp, the size of the empty vector, and faint bands in the correct location for successful ligations.

Fig. 5. PCR of successful ligation. Extremely faint bands are visible in the 500-1000bp range of Samples 2, 3, and 4!

Transformations for the PArsR-spisPink construct were performed following the iGEM transformation protocol. The transformations were screened with colony PCR reactions containing 1x GoTaq Flexi Green Buffer, 0.15mM DNTP, 1.5mM MgCl2, 0.63u GoTaq Polymerase, 0.38μM FWD primer, 0.38μM REV primer, and molecular water to a final volume of 10μL.

Fig. 6. Colony PCR screening. Many colonies were screened to get just 2 positive colonies for the spisPink construct.

In continuation of the amilCP test circuit, we went back to the stocks and digested T7/amilCP with EcoRI-HF and PstI, again following iGEM protocols. After digesting, we ligated the product, this time with a ratio of backbone to insert at 5:1, in an effort to make the reaction more efficient. We screened with our standard PCR protocol and found that the ligation was a success (fig. 7.).

Fig. 7. T7/amilCP ligation screening. Sample 1 shows a band just under 1000bp, the correct size for ligated product.

Week 15

Now that one colony has been confirmed to contain the spisPink construct, we needed to transform the remaining ligations. Transformations followed the usual protocol, and colony screening by PCR.

With the ligation of the amilCP test circuit was complete, we proceeded to transform it according to iGEM protocols. Visual examination of the plates after incubation showed no signs of bacterial color change. We screened the colonies with our PCR protocol and found that the full circuit was present (fig. 8.). This likely means that there was a flaw in circuit design. Members of our team have speculated that this was due to the lack of spaces in the sequence around the promoters and ribosome binding sites.

Fig. 8. T7/amilCP transformation screening. Bands are visible just under 1000bp for Samples 2, 3, and 4. This corresponds to the size of T7/amilCP.

Week 16

After successfully transforming all our individual parts, we finally were getting ready to do minipreps for them. We used the Promega Wizard Plus SV Miniprep Kit and used the protocol provided by Promega to do our minipreps. The whole process did not take too long and soon after, we now had minipreps for pSB1C3/PcArsR, pSB1C3/PArsR-spisPink, and pSB1C3/PArsR-amilCP and stored them in the -20ºC freezer until it was time to send them to iGEM.

At this point, everyone on the team was back in school. Our subteam leaders were starting college and the rest of us were studying for SATs and preparing college applications. Now the team shifts focus to Jamboree!

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-  <body data-spy="scroll" data-target="#parent">
-	<div class="jumbotron">
-		<div class="container">
-			<h1 style="padding: 0px; margin-bottom: 0px;">Notebook</h1>
-		</div>
-	</div>
-	<div class="container">
-		<div class="row">
-			<div class="col-xs-2" id="parent">
-				<ul class="nav nav-pills nav-stacked" data-spy="affix" data-offset-top="275" style="overflow-y: auto; height: 100%">
-					<li class="active" ><a href="#week1">Week 1</a></li>
-					<li><a href="#week2">Week 2</a></li>
-					<li><a href="#week3">Week 3</a></li>
-					<li><a href="#week4">Week 4</a></li>
-					<li><a href="#week5">Week 5</a></li>
-					<li><a href="#week6">Week 6</a></li>
-					<li><a href="#week7">Week 7</a></li>
-					<li><a href="#week8">Week 8</a></li>
-					<li><a href="#week9">Week 9</a></li>
-					<li><a href="#week10">Week 10</a></li>
-					<li><a href="#week11">Week 11</a></li>
-					<li><a href="#week12">Week 12</a></li>
-					<li><a href="#week13">Week 13</a></li>
-					<li><a href="#week14">Week 14</a></li>
-					<li><a href="#week15">Week 15</a></li>
-					<li><a href="#week16">Week 16</a></li>
-					<li><a href="#week17">Week 17</a></li>
-					<li><a href="#week18">Week 18</a></li>
-					<li><a href="#week19">Week 19</a></li>
-				</ul>
-			</div>
-			<div class="col-xs-10">
-			<!-- Wet Lab Overview
-					<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								Our main goals were to 1) clone the three metal transport proteins and pea metallothionein,
-) assemble the transport proteins and metallothionein to make our composite constructs,
-								and 3) monitor sequestration efficiency in our <i>in vivo</i> model using <i>E. coli</i>.
-							</p>
-						</div>
-					</li>
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-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
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-								Text goes here.
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-					</li>
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-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
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-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								Text
-							</p>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Pb_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Lead Subteam</h4>
-									<p>
-										Text
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/a/a9/Hg_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Mercury Subteam</h4>
-									<p>
-										Text
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/dd/Ni_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Nickel Subteam</h4>
-									<p>
-										Text
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/4/40/Cornell_reporters.jpg">
-								<div class="media-body">
-									<h4 class="media heading">Reporters Subteam</h4>
-									<p>
-										Text
-									</p>
-								</div>
-							</div>
-						</div>
-					</li>
-			-->
-<!----------------------------- Week 1 -------------------------------------------->
-				<h3 id="week1">Week 1 (June 2 - June 8)</h3>
-				<hr>
-				<ul class="media-list">
-					<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								Transformation efficiency and protocols were tested - heat shock did not work well, and we came to the conclusion to stick with electroporation.
-The first day of Wet Lab training began on the 7th! We started working on amplification of metallothionein genes - so far nixA and GST-PMT appear to be working.
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-					</li>
-					<li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								Today was the first Dry Lab meeting for the new project! We discussed the mechanics and optimization of general filtration systems. We also started looking for the best way to make hollow fiber reactors the focal point of our filtration system.
-							</p>
-						</div>
-					</li>
-				</ul>
-				<br><br>
-<!----------------------------- Week 2 -------------------------------------------->
-				<h3 id="week2">Week 2 (June 9 - June 15)</h3>
-				<hr>
-				<ul class="media-list">
-					<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								Obtained transformants of pA14F and pC14T heat shocks and both E/B and E/D were successfully heat shocked into DH5a.  Ran into problems later in the week with heat shocked plates from overgrowth and contamination; switched to electroporating of C+F and E+B.
-							</p>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								We began the design process for our filtration system by choosing our ideal target: runoff streams that feed contaminated water into natural rivers. By basing our assumptions around this, we were able to sketch out some functional requirements for our system, such as minimum flow rate, power consumption, materials, etc. We divvied up the system into subparts to research and report back next week.
-							</p>
-						</div>
-					</li>
-				</ul>
-				<br><br>
-<!----------------------------- Week 3 -------------------------------------------->
-				<h3 id="week3">Week 3 (June 16 - June 22)</h3>
-				<hr>
-				<ul class="media-list">
-				<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								This week we ligated various parts (F+A, F+C, E+B, and E+D), transformed, ran colony PCRs, and submitted them for sequencing. Most of our attempts were unsuccessful either due to failed ligations or contamination. We also made T for future minipreps.
-							</p>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								After researching hollow fiber reactors, we found that our assumptions from the last meeting were too liberal. We scaled down all our numbers around a slower flow rate. We discussed our findings on tubing materials, pipe fittings, power sources, and pump options. We sketched out a rough sketch of our system, which included a pump, multiple fiber reactors, larger filters to prevent debris from entering, and a solar panel and battery as power sources.
-							</p>
-						</div>
-					</li>
-				</ul>
-<!----------------------------- Week 4 -------------------------------------------->
-				<h3 id="week4">Week 4 (June 23 - June 29)</h3>
-				<hr>
-				<ul class="media-list">
-				<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								Cloning and troubleshooting continues. E+D, F+C, E+D, E+B4, and E+D11 sequencing results came in negative. The restriction cocktail method was used on F+C and F+A because mRFP kept religating back into the backbone. Plates were successful, but colony PCRs showed they were negative. Repeat cloning. We are facing problems with contamination of lead transporter and R plate.
-Submitting F+C3 for sequencing. We are also working on transforming off the AA and AB kit plate.
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-							<h4 class="media heading">Dry Lab Overview</h4>
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-								After contacting several fiber reactor manufacturers for product quotes, we quickly discovered our hypothetical multi-reactor system was insanely expensive. Since we didn’t want Eric to have to sell an organ to pay for our filtration system, we scaled it down again to a one or two fiber reactor system so it was more feasible.
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-						</div>
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-					</li>
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-				</ul>
+                         top: 1.7em;
+                         font-size: 0.78em;
-<!----------------------------- Week 5 -------------------------------------------->
+                         }
-				<h3 id="week5">Week 5 (June 30 - July 6)</h3>
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-							<h4 class="media heading">Wet Lab Overview</h4>
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-							<p>
+                       }
-								Transformants gave negative signals; many gels lacked proper bands in correct locations.  Growth assay looked good for mercury although concentration needs to be increased for nickel.
+               #teamtext {
-							</p>
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-						<div class="media-body">
+                       }
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								Dan Levine, a Dry Lab member from the 2012 Cornell iGEM team, met with us to help plan our project and pass down ancient Dry Lab design wisdom. He had a unique technique for brainstorming, which was to spend five minutes writing down any ideas you have, regardless of how farfetched, and then share them with the rest of the team. This approach worked extremely well. We had an extensive, positive discussion about our project and decided to completely overhaul our original design. The overall structure of our system was changed to some sort of drum to collect contaminated water, which would then be moved to and filtered in an attached box. We also changed the targeted water source to outflow from a factory, which would fall into the collecting drum.
-							</p>
-						</div>
-					</li>
-				</ul>
-<!----------------------------- Week 6 -------------------------------------------->
-				<h3 id="week6">Week 6 (July 7 - July 13)</h3>
-				<hr>
-				<ul class="media-list">
-				<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								We gathered the data for an assay testing the growth of cells in different heavy metals. In addition, we ligated each the lead transporter (R), and nickel (AA) and mercury (AB) inducible promoters with H. Unfortunately, our transformations did not seem to work for reasons like incomplete digests and inefficient stocks.
-							</p>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								We found a pump deep in the box of Dry Lab components that have accumulated over the years and decide to test it out. We gutted “the Box” from the 2012 team’s project in order to use it as housing for our own filtration system. Unfortunately, the flow was too slow for us to salvage it for our project, but we decided to order new parts in order to begin assembling our own Box.
-							</p>
-						</div>
-					</li>
-				</ul>
-<!----------------------------- Week 7 -------------------------------------------->
-				<h3 id="week7">Week 7 (July 14 - July 20)</h3>
-				<hr>
-				<ul class="media-list">
-				<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								We are troubleshooting some problems with gel purification and smearing on the gel when we visualize it.  Our new stocks of competent cells also appear to be bad. Sequencing of R+H  returned a portion of a random promoter region of Enterococcus, so this will have to be remade. We are in the process of creating many different BioBrick parts, such as AB1+H, F, AC+H,  AB2+H, AC1S+H, AB2S+H, AC+H, AA + H, which are all at various stages of the cloning process.
-							</p>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Pb_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Lead Subteam</h4>
-									<p>
-										R was cultured, miniprepped and quantified. R and H were digested, gel purified and quantified. The pSB1C3 backbone was dephosporylated, and the R and H digests were ligated and transformed.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/a/a9/Hg_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Mercury Subteam</h4>
-									<p>
-										Our first objective is to place the synthesized mercury gene pA14AG into the pSB1C3 backbone. This will be done by digesting pA14AG and pC14H (<i>mRFP</i> in pSB1C3 backbone) with EcoRI and PstI, ligating them, and transforming them. The resulting colonies should contain pA14AG+pC14H (pC14I). pC14I will then be placed upstream of the metallothionein construct pC14AI. This week liquid cultures of pC14H were made from glycerol stock, which were then miniprepped. pA14AG and pC14H were both digested with EcoRI-HF/PstI-HF and gel purified. The gel was good – the pA14AG band was around 800 bp and the pC14H band was around 2000 bp. The concentrations were around 10 ng/μL and they were not worth ligating. Next week we will try re-digesting and re-ligating.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/dd/Ni_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Nickel Subteam</h4>
-									<p>
-										We started out by making glycerol stocks for cultures containing our plasmids pA14AF and pA14AG and miniprepping pA14AF for cloning into the psB1C3 backbone.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/4/40/Cornell_reporters.jpg">
-								<div class="media-body">
-									<h4 class="media heading">Reporters Subteam</h4>
-									<p>
-										The goal of the reporter subteam is to put the reporter (pc14H, which is mRFP) into pc14AA (the nickel inducible promter) and pc14AB (the mercury inducible promoter). Later, we will get a third vector (pc14AC, nixA) that we will also have to put our promoter inside. This week, we made glycerol stocks of pc14AA and pc14AB. We also used glycerol stocks to make cultures of pc14H (mRFP; this is our insert), and later miniprepped these cultures, as well as pre-existing cultures of pc14AA and pc14AB.
-									</p>
-								</div>
-							</div>
-                                                        <div class="media">
-		     <img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/d4/Metallothionein.png">
-								<div class="media-body">
-									<h4 class="media heading">Metallothionein Subteam</h4>
-									<p>
-										This week, AH insert was rehydrated and tranformed, to be prepared in the future.
-									</p>
-								</div>
-							</div>
-						</div>
-					</li>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								We ordered our fiber reactor!
-							</p>
-						</div>
-					</li>
-				</ul>
-<!----------------------------- Week 8 -------------------------------------------->
-				<h3 id="week8">Week 8 (July 21 - July 27)</h3>
-				<hr>
-				<ul class="media-list">
-				<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								We continued working on cloning all of our synthesized parts into the pSB1C3 backbones, despite encountering a number of obstacles such as unsuccessful transformations and low-yielding minipreps and column purifications.
-							</p>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Pb_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Lead Subteam</h4>
-									<p>
-										T was miniprepped. New plates were made. Cultures of the putative transformants were made, but the transformation proved unsuccessful. R and H cultures were grown and miniprepped, but the concentrations were low. New cultures of R and H were subsequently made. A culture of T was made.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/a/a9/Hg_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Mercury Subteam</h4>
-									<p>
-										The double digests of pA14AG and pC14H were redone and new pC14H minipreps made. We increased the amount of DNA used in each digest in hopes of increasing the concentration of the gel purified bands. The gel did not turn out well so we had to re-digest. These newly digested pA14AG and pC14H were run on a gel and gel purified. In the meantime, the gel purified pA14AG and pC14H from last week were ligated (even though the concentrations for each were low) to see if the ligation would work. This ligation was transformed but once plated, there was a lawn of cells – it appears the antibiotic had degraded. The most recent digests turned out to have good concentrations so the backbone was dephosphorylated and ligated with the insert. Transformation and plating of this new ligation produced colonies! Colony cells were grown in liquid cultures, miniprepped, digest screened, and sent to sequencing.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/dd/Ni_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Nickel Subteam</h4>
-									<p>
-										We went through numerous iterations of miniprepping pA14AF and pC14H, digesting both with EcoRI and PstI, gel purifying the insert from pA14AF and backbone from pC14H, dephosphorylating the backbone, ligating, and transforming. By the end of the week, one of the transformations yielded colonies! Here’s hoping…
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/4/40/Cornell_reporters.jpg">
-								<div class="media-body">
-									<h4 class="media heading">Reporters Subteam</h4>
-									<p>
-										This week we almost completed one iteration of the cloning cycle. We digested, cleaned, and ligated pc14AA, pc14AB, and pc14H. We transformed the ligations for a total of 6 plates plates (4 pc14AA +pc14H and 6 pc14B + pc14H). We were able to culture 3 colonies from each of the pc14AA+pc14H plates for a total of 12 cultures, and we miniprepped these cultures for digest screening.
-									</p>
-								</div>
-							</div>
-                                                        <div class="media">
-			<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/d4/Metallothionein.png">
-								<div class="media-body">
-									<h4 class="media heading">Metallothionein Subteam</h4>
-									<p>
-										Miniprepped the cultures made from the colonies of the transformed cells.  After multiple digestion attempts with EcoRI and PstI, we decided to stagger our efforts.  We were going to start running multiple processes of Miniprep/digestion/ligation to increase our potential for success.  By the end of the week, our first ligation samples were ready for heat shocking.  Luckily, the heat shocked contained colonies and we crossed our fingers that these colonies contained was we needed.
-									</p>
-								</div>
-							</div>
-						</div>
-					</li>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								We received most of our ordered parts earlier this week, including the fiber reactor! We tested the fiber reactor with our new pump, and the flow rate was about 0.2 mL/min. After testing the fiber reactor, we brainstormed orientations for our collecting drum and ideas for how to compactly fit the filter and piping into a protected and accessible casing.
-							</p>
-						</div>
-					</li>
-				</ul>
-<!----------------------------- Week 9 -------------------------------------------->
-				<h3 id="week9">Week 9 (July 28 - August 3)</h3>
-				<hr>
-				<ul class="media-list">
-				<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								We have sequence confirmation of our first successful construct: GST-YMT in pSB1C3! Another of our constructs submitted for sequencing was contaminated with that sample, as well, but things are looking hopeful!
-							</p>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Pb_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Lead Subteam</h4>
-									<p>
-										T and S cultures were made and miniprepped. T and R were gel purified. The T digest was dephosphorylated and ligated with R.  The S ligation was transformed.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/a/a9/Hg_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Mercury Subteam</h4>
-									<p>
-										The sequencing did not go well – we think the miniprep might have been contaminated. The minipreps of pC14I were redone and another pA14AG and pC14H ligation was done which we will transform if the sequencing of the new minipreps is again not successful. Sequencing came back as <i>
-GST-YMT</i> which is not what we were hoping for. Thus, we transformed the new ligation and redid the minipreps of pA14AG which were digested with EcoRI-HF/PstI-HF. When loading dye was added to the digested pA14AG prior to running a gel, the digest turned brown…pA14AG was digested again with EcoRI-HF/PstI-HF in two tubes and with EcoRI-HF/SpeI in two tubes but still turned brown when dye was added. We think the EB from the miniprep might have been contaminated and so grew up more pA14AG insert which were miniprepped. The first pA14AG double digest which turned brown was run on a gel. The gel did not look good however, the bands were too long.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/dd/Ni_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Nickel Subteam</h4>
-									<p>
-										This week we grew up, miniprepped, and digest screened a couple colonies from the previous week’s pC14H+pA14AF transformation. The bands on the gel looked to be in the correct location, so off to sequencing they go! <br>
-The rest of the week was spent culturing, miniprepping, and digesting pC14T with EcoRI and SpeI. Our pA14AF EcoRI/SpeI digest will be the insert into the digested pC14T backbone.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/4/40/Cornell_reporters.jpg">
-								<div class="media-body">
-									<h4 class="media heading">Reporters Subteam</h4>
-									<p>
-										Only one of the plates of pc14AB + pc14H had colonies, so we made three cultures and miniprepped them. These minipreps had very low concentrations, so we didn’t digest them, but the minipreps of pc14AA + pc14H from last week had much better concentrations, so we digested them and ran a digest screen. The digest screen was successful, so we submitted a sample for sequencing. The results of the sequencing weren’t what we were looking for, so we cultured a red culture from the plate to prepare for sequencing in order to see if there is a problem with the sequence of the promoter. We miniprepped this RFP culture, and sent it in for sequencing. Meanwhile, we also made overnight ligations of pc14AB + pc14H and transformed them into more cells using heat shock. Unfortunately, nothing grew on the pc14AB + pc14H plates so we threw them out and worked on cleaning and purifying previous pc14AB and pc14H digests.
-									</p>
-								</div>
-							</div>
-                                                        <div class="media">
-			<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/d4/Metallothionein.png">
-								<div class="media-body">
-									<h4 class="media heading">Metallothionein Subteam</h4>
-									<p>
-										The colonies were miniprepped and sent in for sequencing… ‘lo and behold, it worked!  Our construct was a complete success.  The nest construct, AHH, was renamed AI.  Our next task is to digest out the T7 promoter, and then subsequently submitted.  Cultures of AI were made, then miniprepped.  The minipreps were then digested with KpnI to cut out the T7 promoter.  Again, we staggered out multiple processes.  We are hoping to be able to verify a successful digestion next week with a digest screen.
-									</p>
-								</div>
-							</div>
-						</div>
-					</li>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								We decided to collaborate with the Outreach group to create a portfolio of future applications for our filtration system. We had a lot of fun brainstorming cool designs, the collective favorite being the idea of a water roomba. The ideas were distributed among the Dry Lab team and each person was tasked with making a CAD model of theirs.
-							</p>
-						</div>
-					</li>
-				</ul>
-<!----------------------------- Week 10 -------------------------------------------->
-				<h3 id="week10">Week 10 (August 4 - August 10)</h3>
+    .links ul.sub-nav {
-				<hr>
+        /*Length of the text box in the submenu */
-				<ul class="media-list">
+        width: 140px;
-				<li class="media">
+        /*change this to adjust the positions of the boxes from the top */
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
+        margin-top: -42px;
-						<div class="media-body">
+    }
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								We successfully removed the promoter from our GST-YMT biobrick in order to facilitate future applications, and decided to redirect that subteam as an additional force to tackle the cloning of our lead transporter. We’ve also been struggling with the efficacy of our competent cells and it has been holding our cloning efforts back.
-							</p>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Pb_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Lead Subteam</h4>
-									<p>
-										Attempts to transform the ligations continued, without much success.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/a/a9/Hg_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Mercury Subteam</h4>
-									<p>
-										We just realized that the pA14AG culture we had been using was growing in LB without ampicillin, so we grew up another culture overnight, with antibiotic in the LB this time. The pA14AG digested with EcoRI-HF/PstI-HF and with EcoRI-HF/SpeI from last week were run on a gel, the insert bands looked around .7kb so we gel purified. The concentrations were very low – 13 ng/μL for the EcoRI-HF/PstI-HF digests and 5 ng/μL for the EcoRI-HF/SpeI digests. However, the pA14AG digests with EcoRI-HF/PstI-HF were still ligated with pC14H from the metallothionein task force group and transformed. Unfortunately, the plates did not have colonies. pA14AG was again miniprepped and double digested with EcoRI-HF/PstI-HF and EcoRI-HF/SpeI. These digests were run on a gel and gel purified. The gel purified pA14AG digests had good concentrations, were ligated to the pC14H backbone, and transformed/plated. There was one colony on this plate – a LB culture was made of this colony in chloramphenicol and will be miniprepped next week. Four colonies were picked from the plate from earlier this week and LB cultures were made. These will also be miniprepped and sent for sequencing to see if we have successfully transformed pC14I.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/dd/Ni_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Nickel Subteam</h4>
-									<p>
-										Sequencing came back for pC14H+pA14AF. Unfortunately it was not correct, so we needed to go back and reclone those. More cultures of pC14T were miniprepped.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/4/40/Cornell_reporters.jpg">
-								<div class="media-body">
-									<h4 class="media heading">Reporters Subteam</h4>
-									<p>
-										We made ligations from the digests of pc14AB and pc14H – however, we transformed them incorrectly and were unable to plate any cells. Because we ran out of ligations, we made more cultures of pc14AA and pc14AB from the glycerol stocks. We also learned that pc14H is a constitutive promoter, meaning that it will always be on (the cells will always turn red, regardless of whether or not heavy metal is present). Because of this, we started working with a new promoter this week – pc14AJ. The new promoter is a constitutive promoter (amcilCP, and it is blue!). We made a glycerol stock of pc14AJ and cultured from this stock. However, we were unable to continue with the minipreps of pc14AA, pc14AB, and pc14AJ until the beginning of the next week because the lab ran out of supplies.
-									</p>
-								</div>
-							</div>
-                                                        <div class="media">
-			<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/d4/Metallothionein.png">
-								<div class="media-body">
-									<h4 class="media heading">Metallothionein Subteam</h4>
-									<p>
-										AI-T7 was digest screened with SacI, a few of the bands looked good, so they were purified and then sent in for sequencing.  Sequencing came back a success and we had our construct AI-T7, which was renamed AK.  With the quick success of this task force, we were transferred to work on creating the lead construct.  Thus, we started with the same process with R, the lead insert, and H, the psB1C3 backbone.  We miniprepped both pieces this week.
-									</p>
-								</div>
-							</div>
-						</div>
-					</li>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								We made progress on our CAD models.
-							</p>
-						</div>
-					</li>
-				</ul>
-<!----------------------------- Week 11 -------------------------------------------->
+     .links ul.sub-nav li {
-				<h3 id="week11">Week 11 (August 11 - 17)</h3>
+        padding: 1px 10px;
-				<hr>
+    }
-				<ul class="media-list">
-				<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								We are running into more difficulties with mysteriously low-yielding minipreps and unsuccessful transformations, but the cloning subteams march onward!
-							</p>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Pb_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Lead Subteam</h4>
-									<p>
-										The process of reculturing and religating R and T was repeated. The transformation, however, was a failure.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/a/a9/Hg_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Mercury Subteam</h4>
-									<p>
-										pC14I cultures #1 and #3 were miniprepped and a small portion digested with EcoRI-HF/PstI-HF for a digest screen. The pA14AG insert appeared too long so this pC14I is probably not what we want. New pA14AG liquid cultures were made and miniprepped, digested with EcoRI-HF/Psti-HF, and run on a gel which did not turn out well. We had to begin the miniprepping, digesting, and gel purifying process again for pA14AG. Additionlly, new pA14 minipreps were made in case we need them again next week.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/dd/Ni_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Nickel Subteam</h4>
-									<p>
-										After many, many cultures, we were finally able to get a couple of minipreps of pC14T that had good concentration. We proceeded to digest portions with EcoRI/SpeI and EcoRI/PstI for future ligations.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/4/40/Cornell_reporters.jpg">
-								<div class="media-body">
-									<h4 class="media heading">Reporters Subteam</h4>
-									<p>
-										Once the miniprep supplies arrived, we started making <i>16</i> minipreps…which we then lost (on the second-to-last step of the process) to the centrifuge when it refused to open. (Author’s note: as of right now, early October, the centrifuge is still sealed shut and our minipreps are still inside it). The setbacks of the previous week (losing the ligations, having to use a completely different reporter, and the stuck centrifuge) have put the reporter subteam back in square one. We miniprepped 2 cultures of pc14AA, 2 cultures of pc14AB, and 4 cultures of pc14AJ, and then digested, ligated and transformed the ligations. However, once only one plate (pc14AB + pc14AJ) had growth on it, so we made two cultures. Neither of the cultures grew, so nothing could be miniprepped.
-									</p>
-								</div>
-							</div>
-                                                        <div class="media">
-			<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/d4/Metallothionein.png">
-								<div class="media-body">
-									<h4 class="media heading">Metallothionein Subteam</h4>
-									<p>
-										This week comprised not only of struggling to attain successful minipreps for digestions and ligations, but also to hope that the ensuing ligations will successfully have colonies.  After multiple attempts at ligations and transformations, we finally had a single colony that grew that we cultured then digest screened and sent in for sequencing.  Like the previous construct, these are being digested with EcoRI and PstI.
-									</p>
-								</div>
-							</div>
-						</div>
-					</li>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								We did a CT scan of our fiber reactor! We are hoping to do another one after extensive use and compare the two scans.
-							</p>
-						</div>
-					</li>
-				</ul>
-<!----------------------------- Week 12 -------------------------------------------->
-				<h3 id="week12">Week 12 (August 18 - August 24)</h3>
-				<hr>
-				<ul class="media-list">
-				<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								Many team members left for summer vacations, so progress has been a bit slow this week.
-							</p>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Pb_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Lead Subteam</h4>
-									<p>
-										The process of reculturing and religating R and T was repeated. The transformation, however, was a failure.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/a/a9/Hg_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Mercury Subteam</h4>
-									<p>
-										The new pA14AG minipreps were double digested with EcoRI-HF/PstI-HF and gel purified. The gel purified pA14AG from last week was ligated with pC14H which already had been double digested with EcoRI-HF/PstI-HF and dephosphorylated. This ligation was transformed into cells and plated. This process was again repeated with the newly digested pA14AG minipreps from earlier this week. There was one colony that grew on the pC14I plate which was minprepped.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/dd/Ni_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Nickel Subteam</h4>
-									<p>
-										Ligations of pA14AF and pC14T were made multiple times, trying out different combinations of restriction enzymes too (EcoRI/SpeI for both and EcoRI/PstI for both). All were transformed, but colonies only seemed to grow for the EcoRI/PstI digestion enzyme combination. Regardless, colonies were grown to see if it worked.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/4/40/Cornell_reporters.jpg">
-								<div class="media-body">
-									<h4 class="media heading">Reporters Subteam</h4>
-									<p>
-										There was a hiatus this week because all subteam members went back home for a week of summer vacation.
-									</p>
-								</div>
-							</div>
-                                                        <div class="media">
-			<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/d4/Metallothionein.png">
-								<div class="media-body">
-									<h4 class="media heading">Metallothionein Subteam</h4>
-									<p>
-										The sequencing didn’t come back with what we wanted, so we just have to go for a second try.  More rounds of minipreps, more rounds of digestions, more round of failed gel purifications for the insert H.  I’m beginning to sound like a broken record.
-									</p>
-								</div>
-							</div>
-						</div>
-					</li>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								We tested the large filter used for debris with our pump. It was a little difficult because without a battery, we were forced to use the gel box’s power source. Luckily, we still got the water to move completely through the filter.
-							</p>
-						</div>
-					</li>
-				</ul>
-<!----------------------------- Week 13 -------------------------------------------->
-				<h3 id="week13">Week 13 (August 25 - August 31)</h3>
+	}
-				<hr>
-				<ul class="media-list">
-				<li class="media">
+	/* special class that the system uses to make sure the team wants a page to be evaluated */
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
+	.judges-will-not-evaluate {
-						<div class="media-body">
+    	width: 96.6%;
-							<h4 class="media heading">Wet Lab Overview</h4>
+  		margin: 5px 15px;
-							<p>
+  		display: block;
-								Not much progress was made in lead this week.  Mercury sequence-checked their samples to ensure they were correct, proceeding with minipreps of their plasmids.  Nickel and reporters both had issues with digestions and digestion screens.
+		border: 4px solid #3399ff;
-							</p>
+    	font-weight: bold;
+	}
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/a/a9/Hg_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Mercury Subteam</h4>
-									<p>
-										The pC14I miniprep was digested with EcoRI-HF/PstI-HF for a digest screen and run on a gel. pC14I was then sequenced confirmed! This new biobrick consists of mercury <i>MerT and MerP</i> genes, Anderson promoter, and the pSB1C3 backbone. Now our objective is to place pC14I upstream of the metallothionein construct pC14AI and transform the ligated product in BL21 cells. The miniprep of pC14I that was sequence confirmed was transformed into new cells and plated to make glycerol stocks. New LB cultures of pC14AI were grown and were miniprepped.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/dd/Ni_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Nickel Subteam</h4>
-									<p>
-										pC14T+pA14AF colony cultures were miniprepped and digest-screened. Unfortunately, the gel test was inconclusive, so more colonies were cultured, miniprepped, and digest-screened; this second round also led to inconclusive results.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/4/40/Cornell_reporters.jpg">
-								<div class="media-body">
-									<h4 class="media heading">Reporters Subteam</h4>
-									<p>
-										We transformed and plated using ligations of pc14AA + pc14AJ, pc14AB + pc14AJ, and pc14AC +pc14AJ that we’d made prior to leaving for break. Only the pc14AA + pc14AJ plate had any growth on it, despite all the plates being left in the incubator for two days, so we made a culture of this (hoping that it wasn’t contamination that we were culturing). We also cultured some more pc14AA, pc14AB, pc14AC, and pc14AJ from glycerol stocks, and were able to make cleaned digests of the four.
-									</p>
-								</div>
-							</div>
-                                                        <div class="media">
-			<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/d4/Metallothionein.png">
-								<div class="media-body">
-									<h4 class="media heading">Metallothionein Subteam</h4>
-									<p>
-										The sequencing didn’t come back with what we wanted, so we just have to go for a second try.  More rounds of minipreps, more rounds of digestions, more round of failed gel purifications for the insert H.  I’m beginning to sound like a broken record.
-									</p>
-								</div>
-							</div>
-						</div>
-					</li>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								A lot of parts came in this week. We spent the meeting putting the pieces together.
-							</p>
-						</div>
-					</li>
-				</ul>
-<!----------------------------- Week 14 -------------------------------------------->
-				<h3 id="week14">Week 14 (September 1 - September 7)</h3>
-				<hr>
-				<ul class="media-list">
-				<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								With the metallothionein group and the lead group working in tandem, hopefully the lead construct will be completed with haste.  Both nickel and mercury have ligations that are running, so hopefully those will come out strong and with colonies.
-							</p>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Pb_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Lead Subteam</h4>
-									<p>
-										Cultures of R and T were miniprepped. PCR’s of R were run, one with Q5 and one with Phusion. The PCR’s did not show up in a gel. A PCR of varying dilutions of R Miniprep (1 ul, 3 ul, 5 ul, 8ul - each in 200uL H2O) was run. All appeared in a gel. The PCR product was subsequently cleaned.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/a/a9/Hg_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Mercury Subteam</h4>
-									<p>
-										The miniprepped pC14AI was digested with EcoRI-HF/PstI-HF and pC14I digested with EcoRI-HF/SpeI. pC14AI was gel purified, pC14I was dephosphorylated and was ligated to pC14AI. The length of the insert is 1076bp and backbone is about 2761bp. The pC14AI+pC14I ligation was transformed into BL21 cells and plates on chloramphenicol plates. The plates did not show colonies and the ligation was redone and transformed/plated. Liquid cultures were made of the colonies.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/dd/Ni_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Nickel Subteam</h4>
-									<p>
-										We decided to redigest pA14AF and ligate with pC14T, maybe this time it will work? These ligations were then transformed and colonies grown for sequencing. Much to our pleasure, sequencing came back with positive results! We were able to construct pC14K (pC14T+pA14AF)! Glycerol stocks were made of pC14K and pC14H was religated with pA14AF.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/4/40/Cornell_reporters.jpg">
-								<div class="media-body">
-									<h4 class="media heading">Reporters Subteam</h4>
-									<p>
-										We digested the pc14AA + pc14AJ cells and screened them, but the first gel was inconclusive because it was warped, and the second gel showed that the bands were the incorrect lengths (the first, pc14AA was around 2000 base pairs, as it should be, but the second band, pc14AJ was around 1000 bp when it should have been closer to 700 bp). We also made 2 plates of pc14AA + pc14AJ, 3 plates of pc14AB + pc14AJ, and 1 plate of pc14AC + pc14AJ we were able to get 11 cultures from these plates. We miniprepped theses 11 cultures, and we made 12 ligations from cleaned digests left from the previous week. Towards the end of the week we plated a few pc14AA + pc14AJ and pc14AC + pc14AJ transformations.
-									</p>
-								</div>
-							</div>
-                                                        <div class="media">
-			<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/d4/Metallothionein.png">
-								<div class="media-body">
-									<h4 class="media heading">Metallothionein Subteam</h4>
-									<p>
-										Make more cultures of R...Make more cultures of R...Also heat shocked and transformed the ligation from last week, but sadly nothing grew from the colonies.  So we decided to do something new with the transformed colonies and tried colony PCRs, after running the gel of the result, we were rather disappointed (it was like watching an M. Night Shyamalan movie).  The gel was too blurry and unclear have anything conclusive.  So we performed another ligation and hoped that this one would work.
-									</p>
-								</div>
-							</div>
-						</div>
-					</li>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								We bought foam to hold the box’s components in place. We carved out the shapes of the parts into three layers of foam and laid the layers in the box. It was great to see the box finally taking shape.
-							</p>
-						</div>
+</style>
-					</li>
-				</ul>
-<!----------------------------- Week 15 -------------------------------------------->
-				<h3 id="week15">Week 15 (September 8 - September 14)</h3>
-				<hr>
-				<ul class="media-list">
-				<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								No growth shown in lead plate ligations, so we’ll have to try again.  Nickel had some trouble with the digestion screens.  Mercury is still pushing ahead with plenty of minipreps, so hopefully the subsequent digestion/ligation will yield nice results.
-							</p>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Pb_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Lead Subteam</h4>
-									<p>
-										Cultures of T were made from glycerol stock. Quantification of the PCR yielded poor results. The PCR was subsequently restarted. Cultures of T were miniprepped, and the R PCR was redone. The column purification of the R PCR yielded positive results. The R PCR and the T miniprep were subsequently digested. R and T were ligated and transformed. The ligations were plated with chloramphenicol. No growth appeared, so the ligation was repeated.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/a/a9/Hg_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Mercury Subteam</h4>
-									<p>
-										pC14AI+pC14I from the plates was miniprepped and digested with EcoRI-HF/PstI-HF to run a digest screen to see if the ligation was truly of pC14AI and pC14I. The gel showed the insert to be very short and we think it may only be the mercury construct in pSB1C3. The ligation was redone, transformed/plated, and liquid cultures were made of the colonies. pC14I was also miniprepped for future use.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/dd/Ni_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Nickel Subteam</h4>
-									<p>
-										Cultures were made of pC14AI and pC14K for miniprepping and future cloning. pC14AI and pA14AH were digested, but the gel for gel purification came out oddly - bands were not coming out where we would have expected them. We also digested pC14K for more cloning, but none of the digestions had measurable DNA.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/4/40/Cornell_reporters.jpg">
-								<div class="media-body">
-									<h4 class="media heading">Reporters Subteam</h4>
-									<p>
-										We made four cultures from the plates we made last week – 2 of pc14AA + pc14AJ and 2 of pc14AC + pc14AJ. We ran digest screens  on the digested minipreps, but all the bands were the wrong length. There were a few digests remaining from the previous week, so we cleaned, dephosphorylated and ligated them to make 4 ligations (2 of pc14AB + pc14AJ, and 2 of. However, the quality of the ligations is a bit suspect because the digest concentrations were on the lower end. We transformed, plated, and cultured cells using these 4 ligations, but we were unable to miniprep them because the lab ran out of 1.5 mL tubes. We also transformed using a pc14AB + pc14AJ ligation from earlier that we’d found in the box.
-									</p>
-								</div>
-							</div>
-                                                        <div class="media">
-			<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/d4/Metallothionein.png">
-								<div class="media-body">
-									<h4 class="media heading">Metallothionein Subteam</h4>
-									<p>
-										So there was slightly better progress this week, with the overnight ligations yielding a decent number of colonies, with all of the colonies growing in cultures, which was promising.  We tried another colony PCR of the transformants again, but once again, it failed.
-									</p>
-								</div>
-							</div>
-						</div>
-					</li>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								We tried to see the diffusion of luria broth through the fiber reactor. We combined the the LB with tonic water to see the level of fluorescence and qualitatively measure the amount of LB left in the fiber reactor.
-							</p>
-						</div>
-					</li>
-				</ul>
-<!----------------------------- Week 16 -------------------------------------------->
+<!--- THIS IS WHERE THE HTML BEGINS --->
-				<h3 id="week16">Week 16 (September 15 - September 21)</h3>
-				<hr>
-				<ul class="media-list">
-				<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								With tubes arriving, we are blowing full steam ahead.  We have a ligation for nickel, so hopefully this will go through well.  Mercury subteam is making many minipreps to push to finish, so that is looking good…
-							</p>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Pb_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Lead Subteam</h4>
-									<p>
-										Colony growth was observed on the transformant plate. They were cultured, and a colony PCR was run.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/a/a9/Hg_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Mercury Subteam</h4>
-									<p>
-										Many minipreps were made of the pC14AI+pC14I liquid cultures, digested with EcoRI-HF/PstI-HF, and run on a gel. Successful ligation would show a band at around 1.8kb and one of our bands did appear in that area – this band was gel purified and sent for sequencing. In the meantime, pC14I was miniprepped, digested with EcoRI-HF/SpeI, and column purified.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/dd/Ni_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Nickel Subteam</h4>
-									<p>
-										In the process of constructing pC14K+pC14AI, we experienced a bit of difficulty. After multiple failed cloning experiments, we tried synthesizing the part using pC14K+pA14AH. Hopefully the second approach will work!
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/4/40/Cornell_reporters.jpg">
-								<div class="media-body">
-									<h4 class="media heading">Reporters Subteam</h4>
-									<p>
-										Tubes arrived in the lab, so we miniprepped and digested the cultured cells from last week. The gel screen of the digests was <i>textbook perfection</i> for all three of pc14AA + pc14AJ, pc14AB + pc14AJ, and pc14AC + pc14AJ. We sent in samples of the minipreps for sequencing, but the results were strange – sequencing reported that only pc14AJ was in the plasmid, and that none of pc14AA, pc14AB, and pc14AC were present. Luckily earlier in the week, we’d made several ligations of all three that we’d already transformed, plated and cultured so we could still continue with miniprepping these cultures.
-									</p>
-								</div>
-							</div>
-                                                        <div class="media">
-			<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/d4/Metallothionein.png">
-								<div class="media-body">
-									<h4 class="media heading">Metallothionein Subteam</h4>
-									<p>
-										While continuing work on R+H, we received news from high command that were to be repurposed yet again.  This time we are to collect data of metal sequestrations results of e.coli containing the constructs were created.  This requires that we prepare a few glycerol stocks first…
-									</p>
-								</div>
-							</div>
-						</div>
-					</li>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								Tragedy hits the Dry Lab world! We didn’t clean the fiber reactor thoroughly enough after last week’s experiment, resulting in the perfect environment for fungus to grow. We cleaned out the fiber reactor very, very thoroughly with water and put it in the refrigerator to stop the growth of fungus. We will keep an eye out for more fungus.
-							</p>
-						</div>
-					</li>
-				</ul>
-<!----------------------------- Week 17 -------------------------------------------->
-				<h3 id="week17">Week 17 (September 22 - September 28)</h3>
-				<hr>
-				<ul class="media-list">
-				<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								Data collection is just beginning, and by next week hopefully we’ll have some tangible data.  Sequencing came out not well for lead and the digest screens for digestion were inconclusive.  We had some successful ligation in the mercury group, so let’s hope that goes somewhere.
-							</p>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Pb_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Lead Subteam</h4>
-									<p>
-										Gel of colony PCR was run, yielding poor results. Minipreps of some of the cultures were prepared. Colony PCR’s of the R+T cultures were also run. Cultures of relevant strains were remade, and a new colony PCR was run. The cultures of these colonies were miniprepped, and one tube was sent for sequencing. The sequencing yielded unsatisfactory results.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/a/a9/Hg_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Mercury Subteam</h4>
-									<p>
-										The miniprep of pC14AI+pC14I we had sent for sequencing ended up being Neema’s reporter and we think our whole plate was contaminated. We made more pC14AI+pC14I minipreps and digests from the same plate and ran another gel, but we still observed bands in the same region as where Neema’s reporter band was so our hypothesis was virtually confirmed. Thus, we began the entire process of miniprepping and digesting pC14AI and pC14I, gel purifying pC14AI, column purifying and dephosphorylating pC14I, ligating them together and transforming into BL21.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/dd/Ni_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Nickel Subteam</h4>
-									<p>
-										Inserting pA14AH into pC14K did not seem to work judging from the digest-screen. After preparing more pC14AI for cloning, we also were going to try inserting pC14K into pC14AI. However, the gel purification step for that was bizarre and indicated our parts were shorter than what they were. Digest screens of pC14K+pA14AH minipreps also were inconclusive.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/4/40/Cornell_reporters.jpg">
-								<div class="media-body">
-									<h4 class="media heading">Reporters Subteam</h4>
-									<p>
-										This week, we decided to change up our digest protocol a bit – rather than digesting 3000 ng of DNA at a time and incubating the digests for 3 hours, we decided to stick to 1500 ng of DNA and incubate for 1.5 hours maximum because we thought that star activity was causing the DNA to not be digested properly when it was incubated for long periods of time. We made 2 cultures each of pc14AA, pc14AB, pc14AC, and pc14AJ, miniprepped, and digested them for the shorter period of time. When cleaning these digests, however, they smeared in the gel, so we made more cultures of the four plasmids using glycerol stocks. Additionally, we ran digest screens on the cultures from last week, but they were indicative of contamination. By the end of the week, we were able to produce 6 ligations of pc14AC + pc14AJ (we transformed and plated all 6 of these for a total of 12 plates), and we’d started up another batch of cultures from glycerol stocks in case these ligations were not any good. Unfortunately, on the last day of this week, we ran out of Pst1-HF enzyme and were unable to continue with digests because neither of our buffers worked well with both Pst1-HF and Spe1-HF.
-									</p>
-								</div>
-							</div>
-                                                        <div class="media">
-			<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/d4/Metallothionein.png">
-								<div class="media-body">
-									<h4 class="media heading">Metallothionein Subteam</h4>
-									<p>
-										This week, we first had to make chemicompetent glycerol cell stocks of three samples:  AI+R, AI+AF, and AI+AG (AF = nickel construct and AG = mercury construct).  First, we took cells containing the AI construct and transformed the necessary extra plasmids in and created the glyercol stocks.  So we took the AI containing cell and heat shock the necessary plasmids into the cell and made glycerol stocks of those.  Simple.
-									</p>
-								</div>
-							</div>
-						</div>
-					</li>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								We carved out ducts for the piping to go on the top layer of the foam. Also, it looks like the fungus is gone and has not grown anymore. We begin talking about shutting off the pump when there is no water in the collecting drum. We split up into two group to research two main ways of shutting it off: mechanically and electrically.
-							</p>
-						</div>
-					</li>
-				</ul>
-<!----------------------------- Week 18 -------------------------------------------->
-				<h3 id="week18">Week 18 (September 29 - October 5)</h3>
-				<hr>
-				<ul class="media-list">
-				<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								A lot of good data rolled in this week and that is very promising.  Unfortunately PCR cleanups and digestions of were not working well in the nickel and lead subteams, so we will have to push for the next week.
-							</p>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Pb_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Lead Subteam</h4>
-									<p>
-										A digest of an R miniprep was prepared. A PCR of an old R miniprep was run. Cleanup of the PCR yielded poor results, possibly suggesting ethanol contamination.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/a/a9/Hg_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Mercury Subteam</h4>
-									<p>
-										pC14AI digests were run on a gel last week but the ladder did not appear so we could not tell what the length of the insert was (and therefore we could not be sure if the band was what we wanted). Therefore, we began the process again for pC14AI. pC14AI and pC14AK were also miniprepped so they could be turned into iGEM HQ. pC14AI and pC14I were again ligated and transformed into BL21 and plated.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/dd/Ni_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Nickel Subteam</h4>
-									<p>
-										The minipreps of pC14K+pA14AH were sequenced just to see if they were correct. Sadly, they were not - drat! More cultures of pC14K were prepped and quantified. These were then digested for inserting into pC14AI. Here’s hoping the construct works!
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/4/40/Cornell_reporters.jpg">
-								<div class="media-body">
-									<h4 class="media heading">Reporters Subteam</h4>
-									<p>
-										We made a total of 21 digests using a various combination of buffers that we didn’t normally use because Pst1-HF hadn’t arrived yet. Over the course of the week, we made 6 pc14AA + pc14AJ ligations, 6 pc14AB + pc14AJ ligations, and 4 pc14AC + pc14AJ ligations. Transforming using the pc14AC + pc14AJ ligations, we were able to make 4 plates, which yielded 3 successful cultures. We spun down the cultures and stored them in the fridge because the lab had once again run out of tubes. Towards the end of the week, we also made 20 more digests (7 pc14AA, 10 pc14AB, and 3 pc14AC).
-									</p>
-								</div>
-							</div>
-                                                        <div class="media">
-			<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/d4/Metallothionein.png">
-								<div class="media-body">
-									<h4 class="media heading">Metallothionein Subteam</h4>
-									<p>
-										Now the real data collection begins.  Took the three cell types containing AI and a different heavy metal construct and made cultures of them, each with arabinose in them.  After a day of growth, measured OD600 of the cultures then added 1mM of the respective heavy metals into the culture and let grow for another day.  Then spun down cells and retrieved liquid for analysis.  Standard BL21 type cells were used as control as they did not contain any special plasmids.  Much data was collected.
-									</p>
-								</div>
-							</div>
-						</div>
-					</li>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								We drilled holes in the foam for rods meant to hold all the layers together. George also brought the box to the machine shop to drill holes for the rods. After researching both methods of building the water level detector, we decide to focus on the electrical method. The idea is to run a pipe between a phototransistor and an LED. If water is running through the pipe, less light from the LED will reach the phototransistor, and the output current would be less. By measuring the amount of current when there is no water, we can find the shut off threshold for the pump. We will research the specifics of the circuit more for the next meeting.
-							</p>
-						</div>
-					</li>
-				</ul>
-<!----------------------------- Week 19 -------------------------------------------->
-				<h3 id="week19">Week 19 (October 6 - October 12)</h3>
-				<hr>
-				<ul class="media-list">
-				<li class="media">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png">
-						<div class="media-body">
-							<h4 class="media heading">Wet Lab Overview</h4>
-							<p>
-								This is the final stretch, and all of the subteams are running on all four sixes to get as much done as possible.  Unfortunately, the sequencing results just keep coming back as not the correct result, so to work on what we have.
-							</p>
-						  <!-- <div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Pb_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Lead Subteam</h4>
-									<p>
-										Text
-									</p>
-								</div>
-							</div> -->
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/a/a9/Hg_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Mercury Subteam</h4>
-									<p>
-										The remainder of the ligations and new ligations were transformed into BL21 and hopefully these will yield colonies!
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/dd/Ni_small.png">
-								<div class="media-body">
-									<h4 class="media heading">Nickel Subteam</h4>
-									<p>
-										Can anyone sing, “The Final Countdown?” This is it! We iterated through the process of transforming, culturing, miniprepping, and sequencing many times. Sadly, none of the constructs came out correctly and we ran out of time. We put in a great effort, but biology took this round from us.
-									</p>
-								</div>
-							</div>
-							<div class="media">
-								<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/4/40/Cornell_reporters.jpg">
-								<div class="media-body">
-									<h4 class="media heading">Reporters Subteam</h4>
-									<p>
-										Over the course of the weekend, we realized we only had three days to get our final constructs in. Resigning ourselves to the fact that tubes were unlikely to arrive anytime soon, we ligated and transformed using all the digests we had. We’d also had some ligations from previous weeks of pc14AC + pc14AJ that we transformed with early in this final week and sent in for sequencing, after a successful digest screen. Additionally, we finished dephosphorylating the digests we already have to make a second batch of ligations. As our final Hail Mary, we transformed using every single ligation we had, which yielded an astounding 18 plates. Shockingly enough, there was growth on every single plate, which meant we had well over 30 samples to prep so that they could be sent for sequencing. There wasn’t enough time to screen all the samples prior to sequencing, so we selected the samples with the highest concentrations (in the 500 ng/mL range) and sent ten of those in for sequencing. Unfortunately, sequencing results returned exactly what they had in previous times – that only pc14AJ was in the plasmid of the cell. We thought this was because the cell stock had accidentally been made using cells that had pc14AJ as the backbone, and that this problem had been rectified by making new cell stocks, but apparently that was not the case.
-									</p>
-								</div>
-							</div>
-                                                        <div class="media">
-			<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/d/d4/Metallothionein.png">
-							        <div class="media-body">
-									<h4 class="media heading">Metallothionein Subteam</h4>
-									<p> This week, we ran a 12-hour sequestration experiment, taking samples every hour.  The metal concentrations will be analyzed then with a Phen Green test.  We also performed a plate growth assay, recording OD600 of each sample for 24 hours every hour.
-									</p>
-								</div>
-							</div>
-						</div>
-					</li>
-                                             <li class="media dry">
-						<img class="media object pull-left" src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png">
-						<div class="media-body">
-							<h4 class="media heading">Dry Lab Overview</h4>
-							<p>
-								We begin on connecting all the piping and the wiring. We also start to build the circuit for the water level detector.
-							</p>
-						</div>
-					</li>
-				</ul>
-            </div>
-		</div>
-	</div>
-  </body>
+<head>
+	<!-- This tells the browser that your page is responsive -->
+	<meta name="viewport" content="width=device-width, initial-scale=1">
+</head>
+<!-- navbar -->
+<!--- MOBILE NAVBAR HTML --->
+<!--- start of content --->
+<div class="igem_2017_content_wrapper">
+    <head>
+    <style>
+overflow: scroll;
+.button {
+    background-color: #4CAF50;
+    border: none;
+    color: white;
+    padding: 15px 32px;
+    text-align: center;
+    text-decoration: none;
+    display: inline-block;
+    font-size: 16px;
+    margin: 4px 2px;
+    cursor: pointer;
+}
+  /* this is where I can change the background image */
+        #topbox{
+            height:50vh;
+            width: 100%;
+            background-image: url(https://static.igem.org/mediawiki/2018/0/00/T--Tacoma_RAINmakers--TeamMeetingSept.png);
+            background-size:cover;
+            background-attachment: fixed;
+            display: table;
+        }
+        div#topbox h1{
+            font-size: 50px;
+            color: white !important;
+            text-align: center;
+            margin: 0px;
+            display: table-cell;
+            vertical-align: middle;
+            line-height: 50px;
+        }
+        .container{
+  			width:  72%;
+  			margin-left: 15%!important;
+                        margin-top: 2%!important;
+                        display: block
+  		}
+IMG.displayed {
+    display: block;
+    margin-left: auto;
+    margin-right: auto }
+h1{
+color: #204974 !important;
+font-size: 15px;
+text-align: center;
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+color: #9A4E1C !important
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+color: #9A4E1C !important
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+line-height:25px;
+font-family: 'Spinnaker';
+}
+        hr{
+    height: 2px;
+            width: 100%;
+        }
+    </style>
+    </head>
+    <body>
+        <div id="topbox">
+            <h1>Tacoma RAINmakers Lab Notebook</h1>
+        </div>
+<div class="container">
+           <h1>Week 1 </h1> <h2> Digestion and Isolation of pSB1C3 Backbone.</h2>
+           <p> The purpose of digesting the pSB1C3/PArsRGFP construct was to separate the backbone from the former GFP insert. Tacoma RAINmakers sought to isolate the pSB1C3 backbone employed in their 2017 construct, as the GFP reporter complex was no longer desired. Apparent disadvantages of GFP indication in biosensors are ultraviolet readings. The RAINmakers prefered a chromoprotein that produces color in the visible spectrum. Enzymes XbaI and SpeI were used to cleave the terminator sites of the vector, freeing the pSB1C3 backbone. NEB resources confirmed that the Cutsmart Buffer 2.1 is the most compatible with these particular enzymes, providing an optimized environment for digestion. Combining the reagents listed in Table 1.0, the reaction was set at 37ºC in a water bath for 1 hour and 45 minutes. This reaction was completed in duplicate to increase statistical probability of desired backbone DNA. </p>
+            <p>Following completion of pSB1C3 digestion, Tacoma RAINmakers employed a standard procedure listed in the protocol page as “Agarose Gel Electrophoresis.” The purpose of this gel was to confirm if the backbone DNA had been successfully isolated from the undesired GFP insert. As cited in Figure 1, the expected pSB1C3 bands were located at 2000bp. A gel extraction process, also outlined in the protocol section, was completed in order to remove and contain the digested pSB1C3 DNA.
+            </p>
+<p><img src="https://static.igem.org/mediawiki/2018/e/e0/T--Tacoma_RAINmakers--Week1Figure1.png" width="50%" height="50%"><BR><i>Fig. 1. DNA gel of digested parts.</i><p>
+           <h1>Week 2 </h1> <h2> Digestion and Ligation of spisPink, amilCP, and PcArsR Inserts
+    </h2>
+<p>Using IDT stock solutions of chromoprotein and arsenic regulatory DNA, Tacoma RAINmakers completed a standard restriction digest (see protocol page for further information). The notable difference between insert and backbone digestion are the enzymes. Each insert included a SalI enzyme site, which is not compatible with the BioBrick suffix/prefix of the vector. Instead, the SalI site is used to ligate the chromoprotein to PcArsR, rendering the ends of this complete insert as compatible for sticky-end ligation to pSB1C3. After combining reagents listed in Table 2.0, reaction was held in a water bath at 37°C overnight.
+    <p>
+    Once the SalI site in spisPink, amilCP, and PcArsR was successfully sticky-ended, Tacoma RAINmakers were prepared for ligation of each chromoprotein to the arsenic regulator. 60ng of each part were used in order to ensure that there would enough DNA material for the ligation. Having combined the substances from Table 2.1, the reaction was set to ligate overnight at 16°C. Afterwards, SalI was heat inactivated at 80°C, ensuring a complete denature, since the enzyme was no longer required.
+    </p>
+           <h1>Week 3</h1> <h2> Digestion and Ligation of Insert (amilCP/spisPink + PcArsR) to pSB1C3 Backbone</h2>
+<p>Following the ligation of the chromoprotein and PcArsR, the complete insert was digested with enzymes compatible with our pSB1C3 backbone. This process allows sticky-ended ligation in the next step, which increases the chance of a proper insert-backbone ligation. 84 ng of insert DNA was pipetted into the reaction alongside the other reagents mentioned in Table 3.0. The digestion was set at 37°C in a water bath for 1 hour and 30 minutes.</p>
+<p>
+    Tacoma RAINmakers combined the reagents listed in Table 3.1 to ligate the completed insert to the vector. The reaction included a negative control that contained only vector DNA. A notable process involved in ligation reactions is calculating DNA volumes. Typically, a 1:3 ratio of vector to insert ensures that there is a balance of both parts. The RAINmakers employed the NEBioCalculator to determine how many moles were in 1ng of vector (2070bp) and 1ng of insert (1488bp). This calculation translated to 0.8µL of vector and 20µL of insert. The ligation occurred at 16ºC overnight and was heat inactivated at 80ºC for 20 minutes the following morning.
+    </p>
+<h2> Initiation of Individual Chromoprotein and Regulator Plasmid Design</h2>
+    <p>
+    A standard procedure in the Tacoma RAINmakers project is PCR amplification. This process is listed under the protocol page as “Insert PCR Amplification.” In preparation for ligation of inserts (amilCP, spisPink, and PcArsR) into the vector, all insert DNA must be amplified from its original limited stock. Once the PCR reaction has exited the thermocycler, gel electrophoresis must be employed to assess the efficacy of the amplification. As pictured in Figure 3, both the spisPink and amilCP bands successfully appeared at about 1000bp, and the PcArsR expressed at about 550bp. Unfortunately, the PcArsR negative control produced DNA bands, which suggested contamination during the PCR amplification process.
+    </p>
+    <p>
+    Following a successful PCR amplification confirmed by the gel, Tacoma RAINmakers performed a standard gel extraction. With much more insert DNA, RAINmakers were prepared to design three additional plasmids containing single inserts for isolated testing.
+    </p>
+<p><img src="https://static.igem.org/mediawiki/2018/8/82/T--Tacoma_RAINmakers--Week3Figure1.png" width="80%" height="80%"><BR><i>Fig. 2. DNA gel of PCR products.</i><p>
+           <h1>Week 4</h1><h2> Transformation of Arsenic Construct and PCR Screening</h2>
+<p>TThe positive control (pSB1C3 + Insert) and negative control (pSB1C3 only) were transformed using DH5 Alpha Competent E. coli cells. Although the iGEM protocol for transformation states that 1µL of DNA is sufficient, 2µL of DNA were used for both the positive and negative control plates to ensure enough DNA existed for multiple colonies to grow. Transformation reactions were incubated overnight at 37ºC within a 14-18 hour time period. An extended description of the standard transformation process is listed under the protocol page.
+<br>
+The RAINmakers ran a PCR screening for both the positive (vector and insert) and negative (vector only) controls. This PCR allows us to amplify our cloned DNA, which is necessary to do because it will help us determine whether or not the ligation of the vector and insert was successful. The product after our PCR will be used to run a gel, which will help us see which colonies have the vector and insert and which only have the vector. After determining the volumes of the reagents, we decided to add an extra 20% uncertainty to each reagent in the master mix since small volumes of liquid can get stuck in the pipette. We did all these calculations based on the fact that we planned on doing 8 PCR reactions (8 PCR tubes). Before beginning the process of adding all the reagents to our PCR tubes, we needed to determine how long the extension part of PCR should be based on the base pair count of our product. Running a PCR simulation of Snapgene allowed us to find the exact base pair count of our positive control, which is 1597 base pairs. From this, we found that the extension period should be 1 minute and 36 seconds.
+<br>
+After adding all of our reagents together into 8 PCR tubes, we needed to select single colonies from transformation plates from 6/19/18. We determined that 6 of our PCR tubes would have the positive control, 1 would be the negative control and the last tube would have no DNA. Once we selected a single colony and put the DNA into a PCR tube, we streaked it onto a new plate (cut up in 8 different sections for each 8 different colonies) and labeled them 1, 2, 3, 4, 5, 6 (positive control), 7 (nothing) and 8 (negative control). We then incubated this new plate so that more colonies would grow. Finally, we put our PCR tubes in a thermocycler and ran our PCR.
 </p>
-<!--
+    <h2> Blunt-End Digestion and Ligation of pSB1C3 and Inserts</h2>
-NewPP limit report
+<p>
-Preprocessor node count: 9/1000000
+  Designing plasmids with individual inserts becomes slightly complicated, as each insert contains a SalI site that is not compatible with the BioBrick prefix/suffix of the backbone. Hence, Tacoma RAINmakers preferred blunt-end ligation, rendering the incompatible ends irrelevant.
-Post-expand include size: 41/2097152 bytes
+<br>
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+<h1>Week 5 - Week 7</h1>
-		     <li id="t-recentchanges"><a href="/Special:RecentChanges"
+<p>We re-started the cloning process with fresh PCR products and more vector. We re-ligated our pSB1C3 backbone with our PcArsR+SpisPink/PcArsR+amilCP insert several times with only negative results. We decided to re-ligate PcArsR + chromoprotein (spisPink or amilCP), as there were speculations that something went wrong in the initial ligation of the two. This ligation contained 3 uL of PcArsR, 3 uL of chromoprotein (spisPink or amilCP), 1x T4 DNA Ligase Buffer, 1.5U T4 DNA Ligase, and molecular water to a final volume of 10 uL. We ligated at 16ºC overnight, then heat killed at 80ºC for 20 minutes.
-		      title='Recent changes'>Recent changes</a></li>
+<br>
+    <br>
+After this ligation was complete, we digested the newly ligated insert with XbaI and SpeI to get it ready for ligation with the pSB1C3 backbone. This digestion contained 1 uL of Buffer 2.1, 1 uL of XbaI, 1 uL of SpeI, and the newly ligated insert DNA. We did an overnight transformation and then ran PCR on the colonies. To see the results we ran it on a 1% agarose gel and saw that the insert did not ligate to the vector.
-		     <li id="t-whatlinkshere"><a href="/Special:WhatLinksHere/Team:Cornell/notebook"
+This sequence of events repeated itself for many weeks, to great frustration.
-		      title="List of all wiki pages that link here [j]" accesskey="j">What links here</a></li>
-                         <li id="t-recentchangeslinked"><a href="/Special:RecentChangesLinked/Team:Cornell/notebook"
+</p>
-                          title="Recent changes in pages linked from this page [k]" accesskey="k">Related changes</a></li>
+<h1>Week 8</h1><h2>Restart cloning process</h2>
+    <p>After failed attempts to clone the circuit, we decided to start again from the beginning. In the digestion of the backbone, we followed the iGEM protocol, but modified it by doing an overnight digestion at 16ºC instead of a 30 minute digestion. The same digestion was performed on PcArsR, spisPink, and AmilCP. We heat killed the ligase at 80ºC for 20 minutes the next day, and digested the new insert with XbaI and SpeI. After digestion, we did an overnight ligation of the vector and the insert using the slightly modified iGEM protocol with an overnight incubation.
+<BR>
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+<h2>Testing ligation success before transformation</h2>
-	                  title="List of all special pages [q]" accesskey="q">Special pages</a>
+    <p>
-	                 </li>
+The team spent a lot of time transforming without successful insertion of the circuit, and needed a better way to determine the quality of ligation product that was being put into the cells. We reasoned that since PCR can amplify the tiniest amounts of DNA, we may be able to use this to screen the ligation products.<BR>
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+To screen the ligation, we ran end point PCR on the newly ligated parts using our standard PCR protocol. Surprisingly, the PCR confirmed that the vector and insert successfully ligated together! We then transformed our full circuit using the transformation protocol on the iGEM website, and mini-prepped using the Promega Wizard Plus SV Kit. </p>
-	              title="Printable version of this page [p]" accesskey="p">Printable version</a>
+   <img src="https://static.igem.org/mediawiki/2018/b/b6/T--Tacoma_RAINmakers--Week6LigationPCR.jpeg" width="50%" height="50%"><br><p><i>Fig. 3. DNA gel of ligation showing successful ligation.</i><p>
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+<h1>Week 9</h1><h2>Bioengineering Summer Camp</h2>
-	              title="Permanent link to this revision of the page">Permanent link</a>
+<p>The team took a week off from lab work to run a summer bioengineering camp for 9th and 10th grade students. We had a lot of fun teaching the students and used many tools gained from iGEM, like letting students streak out bacteria containing RFP (positive transformation control) and GFP (interlab positive control).</p>
-	             </li>
+<h1>Week 10</h1><h2>Circuit testing</h2>
+<p>We started the process to test the complete arsenic circuit. In vivo testing of full circuits with reporters amilCP and spisPink was performed with 1uM/2uM/10uM concentrations of arsenate/arsenite.
+RAIN's 2017 iGEM team had ran an experiment testing 150uM arsenic which killed the cells during the experiment. A second experiment was conducted where 25uM arsenic was used. This did not kill the cells and GFP was produced. This is the maximum amount of arsenic we want to test.</p>
- 		        <li id="privacy"><a href="/2014.igem.org:Privacy_policy" title="2014.igem.org:Privacy policy">Privacy policy</a></li>
+<p>The limits of detection for our circuit to be viable and to compete with current testing equipment is below 100ppb. At 50ppb soil and water is considered unsafe for consumption or play. At 100ppb the soil or water is deemed worthy for excavation due to its potential health risks. Therefore, we will test 1uM arsenic (equivalent to 75ppb), 2uM arsenic (150ppb) and 10uM arsenic (750ppb).</p>
-		        <li id="disclaimer"><a href="/2014.igem.org:General_disclaimer" title="2014.igem.org:General disclaimer">Disclaimers</a></li>
-	     </ul>
+<p>10mL of liquid culture was created for both spisPink and amilCP the cultures will be set up as follows:<BR>
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+<img src="https://static.igem.org/mediawiki/2018/1/1b/T--Tacoma_RAINmakers--Week10-11TableArsenicTest.png" width="80%" height="80%"><BR></p>
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+<p>The cultures were observed at 1 hour, 3 hours, 24 hours and 48 hours for visual solution color change. During the entirety of the experiment, no color change or production occurred.
-<script>if (window.runOnloadHook) runOnloadHook();</script>
+<h2>Repeat circuit testing with higher concentration of arsenite/arsenate</h2>
+<p>Testing of the amilCP and spisPink in vivo was unsuccessful at 1uM/2uM/10uM. There was also an attempt to grow CFU plates, however, these plates were left in the incubator for two days and overgrew. RAIN's 2017 iGEM team was able to see production of GFP at 25uM concentration of arsenic so this experiment will lie closer to these values.</p>
+<p>Three concentrations or arsenic will be tested this time: 17.65uM (equivalent to 1324ppb), 21.42uM (1606ppb) and 25uM (1875ppb). <BR>
+mL of liquid culture was created for both spisPink and amilCP circuits.</p>
+<p>After growing overnight, there were no color changes in the cultures.</p>
+<h1>Week 11</h1>
+ <h2>More cloning</h2>
+<p>Since we had a lot of trouble seeing any reporter expression, the team wanted to clone the individual parts of just the regulator and just the reporters. This will allow us to see if the reporter will turn on when its not being repressed with ArsR. Unfortunately, we ran into the same problems as before. By this, we mean that the ligation reactions were not demonstrating successful insertion of our DNA into the vector.<BR><BR>
+<h1>Week 12-13</h1>
+<p>Different members of the team kept trying to clone the individual parts. The individual parts were amplified using our standard PCR protocol. Following PCR amplification, the reactions were run at 120V on a 1% Agarose gel. After seeing in the light box that the PCR was successful, the PCR products were extracted using a standard protocol and stored at -20°C.</p>
+   <img src="https://static.igem.org/mediawiki/2018/6/6d/T--Tacoma_RAINmakers--Aug20AnnotatedGel.jpg" width="50%" height="50%"><br><p><i>Fig. 4. DNA gel of PCR products.</i><p>
+The purified parts and pSB1C3 were digested overnight with XbaI and SpeI.
+The ligation was performed the next day using reactions contained 4.1μL of digested insert, 1.35μL digested linear pSB1C3, 1u T4 DNA ligase, and 1x T4 ligase buffer, and Molecular Water to a final volume of 10μL. It was incubated overnight at 16C.
+Then, the ligation was tested with PCR primers amplifying the region between the XbaI and SpeI sites on pSB1C3. The PCR reactions contained 1x GoTaq Flexi Green Buffer, 0.15mM DNTP, 1.5mM MgCl2, 0.63u GoTaq Polymerase, 0.5μL of Ligation Product, 0.38μM FWD primer, 0.38μM REV primer, and molecular water to a final volume of 10μL. We ran it on a 1% agarose gel and saw no bands of the desired size.
+<br><br>
+In order to test the amilCP reporter, we created a circuit with a high expression T7 promoter, amilCP, and the same double terminator used in our other circuits. We began cloning by digesting IDT stock solutions of T7/amilCP with EcoRI-HF and PstI, following iGEM standard protocols. We then ligated T7/amilCP with pSB1C3, using the iGEM ligation protocol. To screen the ligation we used our PCR protocol. After running PCR products on an agarose gel, we found that the ligation failed.
+</p>
+<h1>Week 14</h1>
+    <p>
+We repeated the ligation at 4°C overnight. The reactions contained 5μL digested insert, 5μL digested linear pSB1C3, 1U T4 DNA Ligase, 1x T4 Ligase Buffer, and molecular water to a final volume of 15μL. The ligation was tested in the same way, and once again produced no bands.
+</p>
+    <p>The third attempt at ligation contained 10μL digested insert, 2.5μL of a plasmid with pSB1C3 that we digested using XbaI and SpeI, 1u T4 DNA Ligase, 1x T4 Ligase Buffer, and molecular water to a final volume of 20μL. It was left at room temperature for 1 hour, then moved to incubate at 16°C overnight. The ligation was tested in the same way as the previous 2 attempts. The gel had strong bands at 75bp, the size of the empty vector, and faint bands in the correct location for successful ligations.</p>
+<p><img src="https://static.igem.org/mediawiki/2018/9/96/T--Tacoma_RAINmakers--Sept6AnnotatedGel.jpg" width="75%" height="75%"><br><i>Fig. 5. PCR of successful ligation. Extremely faint bands are visible in the 500-1000bp range of Samples 2, 3, and 4!</i></p>
+<p>Transformations for the PArsR-spisPink construct were performed following the iGEM transformation protocol.
+The transformations were screened with colony PCR reactions containing 1x GoTaq Flexi Green Buffer, 0.15mM DNTP, 1.5mM MgCl2, 0.63u GoTaq Polymerase, 0.38μM FWD primer, 0.38μM REV primer, and molecular water to a final volume of 10μL.</p>
+<p><img src="https://static.igem.org/mediawiki/2018/f/f3/T--Tacoma_RAINmakers--Sept7AnnotatedGel.jpg" width="75%" height="75%"><br><i>Fig. 6. Colony PCR screening. Many colonies were screened to get just 2 positive colonies for the spisPink construct.</i>
+<br><br>
+In continuation of the amilCP test circuit, we went back to the stocks and digested T7/amilCP with EcoRI-HF and PstI, again following iGEM protocols. After digesting, we ligated the product, this time with a ratio of backbone to insert at 5:1, in an effort to make the reaction more efficient. We screened with our standard PCR protocol and found that the ligation was a success (fig. 7.).
+<p><img src="https://static.igem.org/mediawiki/2018/1/1d/T--Tacoma_RAINmakers--MMLigationScreening.jpg" width="45%" height="45%"><br><i>Fig. 7. T7/amilCP ligation screening. Sample 1 shows a band just under 1000bp, the correct size for ligated product.</i></p>
+</p>
+<h1>Week 15</h1>
+    <p>
+Now that one colony has been confirmed to contain the spisPink construct, we needed to transform the remaining ligations. Transformations followed the usual protocol, and colony screening by PCR.
+<br><br>
+With the ligation of the amilCP test circuit was complete, we proceeded to transform it according to iGEM protocols. Visual examination of the plates after incubation showed no signs of bacterial color change. We screened the colonies with our PCR protocol and found that the full circuit was present (fig. 8.). This likely means that there was a flaw in circuit design. Members of our team have speculated that this was due to the lack of spaces in the sequence around the promoters and ribosome binding sites.
+<p><img src="https://static.igem.org/mediawiki/2018/a/ae/T--Tacoma_RAINmakers--MMTransformationScreening.jpg" width="45%" height="45%"><br><i>Fig. 8. T7/amilCP transformation screening. Bands are visible just under 1000bp for Samples 2, 3, and 4. This corresponds to the size of T7/amilCP.</i></p>
+</p>
+<!--╰(•̀ 3 •́)━☆ﾟ.*･｡ﾟbippity-boppity-wiki!-->
+<h1>Week 16</h1>
+    <p>
+After successfully transforming all our individual parts, we finally were getting ready to do minipreps for them. We used the Promega Wizard Plus SV Miniprep Kit and used the protocol provided by Promega to do our minipreps. The whole process did not take too long and soon after, we now had minipreps for pSB1C3/PcArsR, pSB1C3/PArsR-spisPink, and pSB1C3/PArsR-amilCP and stored them in the -20ºC freezer until it was time to send them to iGEM.</p>
+</p>At this point, everyone on the team was back in school. Our subteam leaders were starting college and the rest of us were studying for SATs and preparing college applications. Now the team shifts focus to Jamboree!
+</p>
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