Difference between revisions of "Team:Cadets2Vets/Notebook June"

In an attempt to learn the methods used by other IGEM teams we participated in an inter-team Skype call. We chose to meet with the other teams sponsored by the United States Armed forces because we share many of the same challenges. Our notes were as follows:

Every member attending the Jamboree needs to be able to describe an answer questions about the entire project. It could possibly be a handicap to send people who aren't fully on board with the project.
One of the Navy programmers mentioned some sort of a universal page that affects all of our team pages. Josh, maybe you can look for that and apply some of our sidebar stuff there.
For our presentation, we should go as broad as we can without being boring and then get specific when judges and others ask.
The judges really like lots of pictures and a pretty layout for the poster and powerpoint presentation.
We still need to fill out our InterLab Excel files and go through the protocols.
We need to submit parts. I didn't get the chance to ask how we can check if a part is working, but both teams said we could feel free to email them if we had questions. The Air Force team email is igem@carrollhs.org.
The judges use a VGA cord to attach to computers (one of the bigger ones that needs to be screwed in). No internet is provided, but we could use hotspot if needed.
Conceptual models make good pictures, even if we can only model a piece of our project. There are good poster examples on iGEM.
For our procedures on our presentation and poster, we should include project-specific protocols (e.g. the lyophilization process or testing for GFP) before anything more basic like PCR, but it might be good to specify our specific reagents and primers used for more common things.
A lot of projects don't end up working, but some teams cheat and do a project for multiple years in order to make it work, or they stack a team with a ridiculous number of people. It seems judges might (?) be a little bit lenient with those who fudge the rules.
The Carroll College team has already gone through the InterLab procedures.
We need to specify our part for the GFP we're using.
For best human practices integration, a lot of teams go all out and serve food or draw pictures with GFP, so we need to do similar if we want to be competitive. Could we incorporate the Science Breakfasts somehow? We could also practice talking about our project to people at these events.

With the help of high school interns Keshava Katti, Kian Croston, and Josh Grace, senior technician Ryan Brisban performed multiple quantitative and qualitative protein assays to assess the presence of GFP in situ. To these ends, 8 circuit samples were put in a Light Cycler to incubate for 8 hours at 37 degrees Celsius. 4 samples were run with no Arsenic regulator present, meaning that GFP should have been produced continuously for the entire 8 hour period. 3 were run as controls with only broth and no circuit, and 1 control of pure molecular water was run as a baseline measurement. After 8 hours, the normalized fluorescence of the 4 samples containing GFP was significantly higher than any of the controls. To supplement these findings, the Cadets2Vets team ran an acrylamide protein gel on the samples, and although the gel broke in processing, there appeared to be a protein band corresponding to GFP’s known molecular size in kilo-Daltons. A second acrylamide protein gel assay is currently being run to increase the number of trials and provide better evidence than the broken gel. A second assay was also run using Qubit’s total protein assay. All samples were spec’d after creating a linear model of RFUs using standards, but they exceeded the range of the sensor, and thus needed to be diluted. After dilution, the protein concentration was determined to be in the ~1000s of ng/µL for each of the seven samples containing broth, whereas the sample containing molecular water exhibited control values too low for the Qubit to read. The 4 samples containing no Arsenic regulator with the circuit appeared to have lower protein concentrations by ~100-200 ng/ µL than the 3 samples containing only reagents and water. This finding was unexpected to the Cadets2Vets team, who hope that further analysis and measurement will determine the source of the extra protein. The current hypothesis is that the samples containing our circuit functionalized more of the reactants from the reaction matrix.

On July 19, 2017, senior technician Ryan Brisban with the help of high school interns Keshava Katti, Kian Croston, and Josh Grace analyzed a second acrylamide protein assay. This was done in order to qualitatively demonstrate the presence of GFP in situ for Cadets2Vets’ iGEM arsenic-sensing circuit, and increase the power of the evidence collected from the previous acrylamide protein assay whose gel broke. The second acrylamide protein gel assay presents strong qualitative evidence for the presence of GFP and known regulatory proteins in situ. The fact that the Cadets2Vets team’s regulated plasmids appear to have a high degree of variability (Fig 1.1) and protein smearing leads the team to believe that a shielding mechanism is occurring where the excess regulatory protein in situ prevents GFP from presenting as strongly as it otherwise could. The Cadets2Vets team plans to next perform ticketing and brainstorm methods to increase GFP signaling efficiency.

Fig 1.1 – Acrylamide protein gel assay. First ladder (far left) is a standard. Second, third, and fourth are reaction replicates of an unregulated GFP promoter complex, with two strongly visible bands (bottom of latter bands demonstrate presence of reaction proteins at 13.3 kilo-Daltons; top of ladder bands demonstrate presence of GFP at 32.7 kilo-Daltons). The fifth ladder is a reaction control with no plasmid (bottom band presenting for reaction proteins). The sixth, seventh, and eighth ladders contain reaction replicates of the Cadets2Vets team’s regulated GFP plasmid. Variability demonstrated is due to varying amounts of regulatory protein and variability.

On July 20, 2017, senior technician Ryan Brisban with the help of high school interns Keshava Katti, Kian Croston, and Josh Grace created tickets by aliquoting Ars 1.1 circuits into wells with various other reagents. This was done to test the in-paper efficacy of the circuits that the Cadets2Vets team engineered.

To these ends, the Cadets2Vets team aliquoted their circuit and reaction solution without arsenic ions to each well of the negative control. The Cadets2Vets team aliquoted their circuit and reaction solution with arsenic ions into each well of the positive control.

The reaction solution contained 7.5 S30 Extract, 10 S30 Promega, 2.5 amino (+), 0.5 amino (-), and 2 molecular water. For the positive test, 2.5 of our circuit plasmid and machinery was added, and the 2 molecular water contained arsenite and arsenate at a 150 concentration. 2 of water (molecular for negative control, arsenic ion and water mixture for positive test) was added to wells in column 2, and 4 of water (molecular for negative control, arsenic ion and water mixture for positive test) was added to wells in column 3. The wells in the second column had an arsenic ion concentration of 82.5 and the wells in the third column has an arsenic ion concentration of 208.3 for the positive test ticket.

These tickets were then let to incubate overnight (~18 hours) at ~40 degrees Celsius, at which point they were analyzed qualitatively using a backlight (Fig 1.1). Although measures were taken to try to prevent the tickets from drying up by supplying a source of humidity in the petri dishes, the Negative Control Paper Circuit unfortunately dried out slightly, which is why some of the wells seem lighter than the others. Despite this, no fluorescence was detected on any of the negative control wells. The positive test exhibited fluorescence in the first column of wells, but the second and third wells exhibited no fluorescence. The Cadets2Vets team currently hypothesizes that this may be a result of arsenic ions saturating the pores of the ticket, inhibiting the function of the cellular machinery by destabilizing GFP conformation, or negatively influencing ligand effects, preventing the production of GFP.

Moving forward, the Cadets2Vets team plans to amplify the signal of the positive test by spiking in more regulatory circuits to the abiotic wells. The team hypothesizes that this allow more of the arsenic ions to be functionalized by the regulatory proteins to produce GFP, increasing the ratio of GFP created. The Cadets2Vets team also plans to alter the percentage-based concentrations of arsenic in the positive test wells to determine the current upper abiotic LOD. Lastly, the Cadets2Vets team will test new methods to keep the tickets from drying out during incubation to find a (hopefully) more effective method than what is currently being used.

On July 25, 2017, senior technician Ryan Brisban with the help of high school interns Keshava Katti, Kian Croston, and Josh Grace ran a second ticket assay to increase the power of the previous test and provide clearer results.

To these ends, the Cadets2Vets team aliquoted their circuit and reaction solution without arsenic ions to each well of the negative control. The Cadets2Vets team aliquoted their circuit and reaction solution with arsenic ions into each well of the positive control.

The reaction solution contained 7.5 LS30 Extract, 10 LS30 Promega, 2.5 Lamino (+), 0.5 Lamino (-), and 2 Lmolecular water. For the positive test, 2.5 Lof our circuit plasmid and machinery was added, and the 2 Lmolecular water contained arsenite and arsenate at a 150 M concentration. The negative control ticket used only 2 Lof reaction mixture for the first two columns of wells on the left, and the other two columns of wells were filled with 2 Lof molecular water. The positive test ticket used 2 Lof the test mixture in the first column of wells, and 4Lof test mixture in the second column of wells. The other two columns of wells were filled with 2Lof molecular water.

These tickets were then let to incubate overnight (~18 hours) at ~40 degrees Celsius, at which point they were analyzed qualitatively using a backlight (Fig 1.1 and Fig 1.2). Once again, no fluorescence was detected on any of the negative control wells. The positive test exhibited fluorescence in the first and second columns of wells, and qualitatively it appears that there may have been some form of dose response, as the second column of wells appears brighter. The negative control appears to have dried again slightly, and the Cadets2Vets team will be aliquoting more reaction mixture into each well during later tests to try and combat this issue.

Difference between revisions of "Team:Cadets2Vets/Notebook June"

Revision as of 22:18, 1 August 2017

Our Progress

Notebook

JULY 14, 2017

INTER-TEAM MEETING

JULY 18, 2017

JULY 20, 2017

July 20, 2017

July 25, 0217

Image Gallery

In this chart we can clearly see Positive Controls 1 and 2 expressing fluorescent signatures

Initial text of circuit in the presence of 25 um arsenite and arsenate solution we were able to generate fluorescent signature.

Follow us on Social Media!

2304 Jefferson Ave, Tacoma, WA 98402

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-			<a class="removable-parent" href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-6b444095-lok1anrm" data-link-type="EXISTING"   target="_self" >
+			<a class="removable-parent" href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-466a74f8-j2jzxizg" data-link-type="EXISTING"   target="_self" >
-			<span id="9baf11fa"  class="preview-element Link item-link magic-circle-holder text-element custom" data-menu-name="PREVIEW_LINK" >Notebook</span>
+			<span id="b951bd16"  class="preview-element Link item-link magic-circle-holder text-element custom" data-menu-name="PREVIEW_LINK" >Notebook</span>
+			</a>
+		</li>
+		<li class="removable-parent">
+			<a class="removable-parent" href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-5515b55f-jgjhhkan" data-link-type="EXISTING"   target="_self" >
+			<span id="928ba103"  class="preview-element Link item-link magic-circle-holder text-element custom" data-menu-name="PREVIEW_LINK" >Safety</span>
+			</a>
+		</li>
+		<li class="removable-parent">
+			<a class="removable-parent" href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-9e25704e-ynxs9cit" data-link-type="EXISTING"   target="_self" >
+			<span id="e08666cf"  class="preview-element Link item-link magic-circle-holder text-element custom" data-menu-name="PREVIEW_LINK" >Notebook-July</span>
+			</a>
+		</li>
+		<li class="removable-parent">
+			<a class="removable-parent" href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-59a5b-xtfijafb" data-link-type="EXISTING"   target="_self" >
+			<span id="bd841f1b"  class="preview-element Link item-link magic-circle-holder text-element custom" data-menu-name="PREVIEW_LINK" >Parts</span>
+			</a>
+		</li>
+		<li class="removable-parent">
+			<a class="removable-parent" href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-04d15a56-3o81aqko" data-link-type="EXISTING"   target="_self" >
+			<span id="a6cf4805"  class="preview-element Link item-link magic-circle-holder text-element custom" data-menu-name="PREVIEW_LINK" >Description</span>
 			</a>
@@ Line 173: / Line 225: @@
 			<a class="removable-parent" href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-4f279020-pcxhuaas" data-link-type="EXISTING"   target="_self" >
-			<span id="23b411c4"  class="preview-element Link item-link magic-circle-holder text-element custom" data-menu-name="PREVIEW_LINK" >Team Members</span>
+			<span id="element-077b707f6118c36"  class="preview-element Link item-link magic-circle-holder text-element custom" data-menu-name="PREVIEW_LINK" >Team</span>
+			</a>
+		</li>
+		<li class="removable-parent">
+			<a class="removable-parent" href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-51653ffc-lok1anrm" data-link-type="EXISTING"   target="_self" >
+			<span id="2ed4772d"  class="preview-element Link item-link magic-circle-holder text-element custom" data-menu-name="PREVIEW_LINK" >Notebook-July</span>
+			</a>
+		</li>
+		<li class="removable-parent">
+			<a class="removable-parent" href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-fed51ac9-l8qwmrzh" data-link-type="EXISTING"   target="_self" >
+			<span id="ece571a0"  class="preview-element Link item-link magic-circle-holder text-element custom" data-menu-name="PREVIEW_LINK" >Collaborations</span>
+			</a>
+		</li>
+		<li class="removable-parent">
+			<a class="removable-parent" href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-ab048c8e-h2ujhr1w" data-link-type="EXISTING"   target="_self" >
+			<span id="463d44e3"  class="preview-element Link item-link magic-circle-holder text-element custom" data-menu-name="PREVIEW_LINK" >Experiments</span>
+			</a>
+		</li>
+		<li class="removable-parent">
+			<a class="removable-parent" href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-ae64641e-hata8o9c" data-link-type="EXISTING"   target="_self" >
+			<span id="ae4d1fb8"  class="preview-element Link item-link magic-circle-holder text-element custom" data-menu-name="PREVIEW_LINK" >Design</span>
 			</a>
@@ Line 201: / Line 297: @@
 		data-MENU_SCROLL="false"
 		data-ALWAYS_MINIFIED="false"
-		data-MENU_POSITION="top"
+		data-MENU_POSITION="none"
 		data-MENU_ALIGN="left"
 		data-BACKGROUND_COLOR="rgb(124, 100, 41)"
@@ Line 214: / Line 310: @@
-	<!-- SERVICES START -->
+	<!-- PROJECTS START -->
-		<div  id="vbid-a18d59f0-36ajwbux"  class="master item-box  gallery-box style-a18d59f0-9hy0h3os    button-effects btn_hover3    custom  " data-holder-type="gallery"" data-holder-type="gallery"  data-child-type="NATIVE_ORDER"  data-styleid="style-a18d59f0-9hy0h3os" data-preview-styleid='style-a18d59f0-9hy0h3os' data-preset-type-id="SERVICES">
+		<div  id="vbid-0e6009b3-5tjceeyo"  class="master item-box  gallery-box style-0e6009b3-uz8lja45    button-effects btn_hover3 items-hover-effects item_hover11     " data-holder-type="gallery"" data-holder-type="gallery"  data-child-type="NATIVE_ORDER"  data-styleid="style-0e6009b3-uz8lja45" data-preview-styleid='style-0e6009b3-uz8lja45' data-preset-type-id="PROJECTS">
 			<div id="no-image" class="stripe-background load-high-res " ></div>
-			<div class="gallery-wrapper item-wrapper services-wrapper ">
+			<div class="gallery-wrapper item-wrapper projects-wrapper ">
-					<!-- CACHED VERSION (7) OF vbid-a18d59f0-36ajwbux FROM  17-07-2017 23:47:19 --><div  class="sub container style-a18d59f0-9hy0h3os content matrix  custom " data-itemtype="folder" data-creator="" data-itemname="LOADING"  data-itemslug="loading" data-itemstyleid="style-a18d59f0-9hy0h3os" data-margintop="" data-arranger="matrix" data-layout="multi" data-vbid="vbid-a18d59f0-36ajwbux" data-preset-type-id="SERVICES" data-preview-style="style-a18d59f0-9hy0h3os" data-style="style-8ec4c-t1avbp6mmi" data-absolute-path="https://xprs.imcreator.com/viewer"  >
+					<!-- CACHED VERSION (7) OF vbid-0e6009b3-5tjceeyo FROM  01-08-2017 17:35:38 --><div  class="sub container style-0e6009b3-uz8lja45 content matrix   " data-itemtype="folder" data-creator="" data-itemname="LOADING"  data-itemslug="loading" data-itemstyleid="style-0e6009b3-uz8lja45" data-margintop="" data-arranger="matrix" data-layout="multi" data-vbid="vbid-0e6009b3-5tjceeyo" data-preset-type-id="PROJECTS" data-preview-style="style-0e6009b3-uz8lja45" data-style="style-8ec4c-t1avbp6mmi" data-absolute-path="/viewer"  >
 	<!-- ARRANGER SETTINGS -->
 	<div class="arranger-settings" style="display:none;"
 		data-ARRANGER_TYPE="matrix"
-		data-ARRANGER_COLS="1"
+		data-ARRANGER_COLS="2"
 		data-ARRANGER_ITEM_MAX_WIDTH="1000"
-		data-ARRANGER_ITEM_MIN_WIDTH="699.3"
+		data-ARRANGER_ITEM_MIN_WIDTH="343.7"
-		data-ARRANGER_ITEM_RATIO="0.4"
+		data-ARRANGER_ITEM_RATIO="0.5"
-		data-ARRANGER_ITEM_SPACING="10"
+		data-ARRANGER_ITEM_SPACING="8"
 		data-ARRANGER_ITEMS_PER_PAGE="all"
 		data-ARRANGER_ORDER_TYPE="regular"
@@ Line 240: / Line 336: @@
 		<!-- STRIPE HEADER -->
 		<div class="stripe-header-wrapper">
-			<div  id="vbid-a18d59f0-hoiablr0"  class="stripe-header sub item-box page-box style-a18d59f0-9hy0h3os       " data-holder-type="page"" data-holder-type="page"  data-child-type="STYLE"  data-styleid="style-a18d59f0-9hy0h3os" data-preview-styleid='style-a18d59f0-9hy0h3os' data-preset-type-id="UNRESOLVED">
+			<div  id="vbid-0e6009b3-f4joueoo"  class="stripe-header sub item-box page-box style-0e6009b3-uz8lja45       " data-holder-type="page"" data-holder-type="page"  data-child-type="STYLE"  data-styleid="style-0e6009b3-uz8lja45" data-preview-styleid='style-0e6009b3-uz8lja45' data-preset-type-id="UNRESOLVED">
 				<div class="page-wrapper item-wrapper ">
-					<div class="item-content leaf blocks_layout page content" data-self="vbid-a18d59f0-hoiablr0" data-preview-style="style-a18d59f0-9hy0h3os" data-style="style-8ec4c-ed91kjv1sl" data-orig-thumb-height="" data-orig-thumb-width="" data-vbid="vbid-a18d59f0-hoiablr0" data-bgimg="">
+					<div class="item-content leaf blocks_layout page content" data-self="vbid-0e6009b3-f4joueoo" data-preview-style="style-0e6009b3-uz8lja45" data-style="style-8ec4c-t1avbp6mmi" data-orig-thumb-height="" data-orig-thumb-width="" data-vbid="vbid-0e6009b3-f4joueoo" data-bgimg="">
 			<div class="helper-div middle" >
 			<!-- <div class="benet" style="min-height:inherit;"></div> -->
@@ Line 256: / Line 352: @@
 <div class="blocks-preview-title-holder removable-parent order-handle">
-	<h2 id="vbid-a18d59f0-y55gbg2o" class="preview-element blocks-preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="BLOCKS_PREVIEW_TITLE" >Our progress</h2>
+	<h2 id="vbid-0e6009b3-zw9rdw1k" class="preview-element blocks-preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="BLOCKS_PREVIEW_TITLE" >Our Progress</h2>
 </div>
@@ Line 286: / Line 382: @@
-		<div  id="vbid-7f31816d-kdwtqku4"  class="sub item-box  page-box style-a18d59f0-9hy0h3os          " data-holder-type="page"" data-holder-type="page"  data-child-type="STYLE"  data-styleid="style-a18d59f0-9hy0h3os" data-preview-styleid='style-a18d59f0-9hy0h3os' data-preset-type-id="UNRESOLVED">
+		<div  id="vbid-0e6009b3-2o9cdajh"  class="sub item-box  page-box style-0e6009b3-uz8lja45          " data-holder-type="page"" data-holder-type="page"  data-child-type="STYLE"  data-styleid="style-0e6009b3-uz8lja45" data-preview-styleid='style-0e6009b3-uz8lja45' data-preset-type-id="UNRESOLVED">
 			<div class="page-wrapper item-wrapper ">
-					<div class="item-content leaf multi_layout page content -container" data-self="vbid-7f31816d-kdwtqku4" data-preview-style="style-a18d59f0-9hy0h3os" data-style="style-8ec4c-ed91kjv1sl" data-orig-thumb-height="3227" data-orig-thumb-width="4840" data-vbid="vbid-7f31816d-kdwtqku4" data-bgimg="https://lh3.googleusercontent.com/ZSt9Gn-cEkmwhzEXFn3cbjuLsTft1FcgdEzYfsi2zWGPdbTWHckKzUs5QWX-MvTVtV1Qa6Gy3jTpWtjzybE">
+					<div class="item-content leaf multi_layout page content -container" data-self="vbid-0e6009b3-2o9cdajh" data-preview-style="style-0e6009b3-uz8lja45" data-style="style-8ec4c-ed91kjv1sl" data-orig-thumb-height="360" data-orig-thumb-width="640" data-vbid="vbid-0e6009b3-2o9cdajh" data-bgimg="https://lh3.googleusercontent.com/HVaw8FL-ORNXLdBIbZDobnN2gpvHf144FRH_zNZHawWKWmLz24O8CrxpCxGgI0bkFt6jEMGhGMP0W8A-dQI">
 <div  class="multi-container preview image-cover" >
 	<div class="Picture item-preview">
@@ Line 296: / Line 392: @@
 			<div id="no-image" class="background-image-div preview-element image-source magic-circle-holder unfold-left load-high-res" data-menu-name="BACKGROUND_IMAGE" style="">
 			</div>
-			<div class="helper-div middle-right" >
+			<div class="helper-div middle-center" >
 			<!-- <div class="benet" style="min-height:inherit;"></div> -->
 			<div class="pic-side">
 				<div class="vertical-aligner">
-					<div id="vbid-7f31816d-sj362pjg-holder"  class="preview-image-holder inner-pic-holder" data-menu-name="PREVIEW_INLINE_IMAGE_HOLDER">
+					<div id="vbid-0e6009b3-wytastvw-holder"  class="preview-image-holder inner-pic-holder" data-menu-name="PREVIEW_INLINE_IMAGE_HOLDER">
-	<a class="image-link top-layer not-wrapping" href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-6b444095-lok1anrm"  data-link-type="EXISTING" target="_self" ></a>
+	<div  id="vbid-0e6009b3-wytastvw" class="inner-pic preview-element  magic-circle-holder  load-high-res " data-menu-name="PREVIEW_INLINE_IMAGE" style="background-image:url(https://lh3.googleusercontent.com/HVaw8FL-ORNXLdBIbZDobnN2gpvHf144FRH_zNZHawWKWmLz24O8CrxpCxGgI0bkFt6jEMGhGMP0W8A-dQI=s300);"  data-orig-width="640" data-orig-height="360" >
-	<div  id="vbid-7f31816d-sj362pjg" class="inner-pic preview-element  magic-circle-holder  load-high-res " data-menu-name="PREVIEW_INLINE_IMAGE" style="background-image:url(https://lh3.googleusercontent.com/ZSt9Gn-cEkmwhzEXFn3cbjuLsTft1FcgdEzYfsi2zWGPdbTWHckKzUs5QWX-MvTVtV1Qa6Gy3jTpWtjzybE=s300);"  data-orig-width="4840" data-orig-height="3227" >
 		<!-- VIDEO TEMPLATE -->
@@ Line 340: / Line 434: @@
-<br class="upper-line-break" />
+<div class="element-placeholder" data-elementtype='TITLE' style="display:none;"></div>
-<div class="preview-title-holder removable-parent order-handle">
-		<a href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-6b444095-lok1anrm"  data-link-type="EXISTING" target="_self" >
+<div class="element-placeholder" data-elementtype='DIVIDER' style="display:none;"></div>
-	<h2 id="vbid-7f31816d-gjyccn16" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >Overview<br></h2>
-	</a>
-</div>
-<br class="lower-line-break" />
@@ Line 355: / Line 445: @@
 <div class="element-placeholder" data-elementtype='SUBTITLE' style="display:none;"></div>
-<div class="element-placeholder" data-elementtype='DIVIDER' style="display:none;"></div>
@@ Line 376: / Line 462: @@
 											<div class="preview-item-links order-handle ">
 	<div class="preview-links-wrapper">
+		<span id="vbid-0e6009b3-dppprmzs"  class="preview-element Link item-link magic-circle-holder text-element " data-menu-name="PREVIEW_LINK" >Project No 1</span>
 <div class="element-placeholder" data-elementtype='LINK' style="display:none;"></div>
@@ Line 410: / Line 500: @@
-		<div  id="vbid-a18d59f0-clydsvwt"  class="sub item-box  page-box style-a18d59f0-9hy0h3os          " data-holder-type="page"" data-holder-type="page"  data-child-type="STYLE"  data-styleid="style-a18d59f0-9hy0h3os" data-preview-styleid='style-a18d59f0-9hy0h3os' data-preset-type-id="UNRESOLVED">
+		<div  id="vbid-0e6009b3-dk9l6lqv"  class="sub item-box  page-box style-0e6009b3-uz8lja45          " data-holder-type="page"" data-holder-type="page"  data-child-type="STYLE"  data-styleid="style-0e6009b3-uz8lja45" data-preview-styleid='style-0e6009b3-uz8lja45' data-preset-type-id="UNRESOLVED">
 			<div class="page-wrapper item-wrapper ">
-					<div class="item-content leaf multi_layout page content -container" data-self="vbid-a18d59f0-clydsvwt" data-preview-style="style-a18d59f0-9hy0h3os" data-style="style-8ec4c-ed91kjv1sl" data-orig-thumb-height="1280" data-orig-thumb-width="1920" data-vbid="vbid-a18d59f0-clydsvwt" data-bgimg="https://lh3.googleusercontent.com/ApBhfyeCvgHc8cpLSPPdB9YD6xhZca0o3tLl7Ar_GxtX2YNNXIPpyvKCvl40IKjZZ55CPAqyKieTH45gtx8">
+					<div class="item-content leaf multi_layout page content -container" data-self="vbid-0e6009b3-dk9l6lqv" data-preview-style="style-0e6009b3-uz8lja45" data-style="style-8ec4c-ed91kjv1sl" data-orig-thumb-height="697" data-orig-thumb-width="1000" data-vbid="vbid-0e6009b3-dk9l6lqv" data-bgimg="https://lh3.googleusercontent.com/4CefoTb07DPKeJgwA8neHS1bWCV13lCqXZd52jQ-hnggf2vQYEU_JMcj12TaJZr5kqXMKznM55L0fR43">
 <div  class="multi-container preview image-cover" >
 	<div class="Picture item-preview">
@@ Line 420: / Line 510: @@
 			<div id="no-image" class="background-image-div preview-element image-source magic-circle-holder unfold-left load-high-res" data-menu-name="BACKGROUND_IMAGE" style="">
 			</div>
-			<div class="helper-div middle-right" >
+			<div class="helper-div middle-center" >
 			<!-- <div class="benet" style="min-height:inherit;"></div> -->
 			<div class="pic-side">
 				<div class="vertical-aligner">
-					<div id="vbid-a18d59f0-mexiqjlg-holder"  class="preview-image-holder inner-pic-holder" data-menu-name="PREVIEW_INLINE_IMAGE_HOLDER">
+					<div id="vbid-0e6009b3-ytsnbyiy-holder"  class="preview-image-holder inner-pic-holder" data-menu-name="PREVIEW_INLINE_IMAGE_HOLDER">
-	<a class="image-link top-layer not-wrapping" href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-5f09d343-lok1anrm"  data-link-type="EXISTING" target="_self" ></a>
+	<a class="image-link top-layer not-wrapping" href="https://2017.igem.org/Team:Cadets2Vets/Notebook_June"  data-link-type="EXTERNAL" target="_blank" ></a>
-	<div  id="vbid-a18d59f0-mexiqjlg" class="inner-pic preview-element  magic-circle-holder  load-high-res " data-menu-name="PREVIEW_INLINE_IMAGE" style="background-image:url(https://lh3.googleusercontent.com/ApBhfyeCvgHc8cpLSPPdB9YD6xhZca0o3tLl7Ar_GxtX2YNNXIPpyvKCvl40IKjZZ55CPAqyKieTH45gtx8=s300);"  data-orig-width="1920" data-orig-height="1280" >
+	<div  id="vbid-0e6009b3-ytsnbyiy" class="inner-pic preview-element  magic-circle-holder  load-high-res " data-menu-name="PREVIEW_INLINE_IMAGE" style="background-image:url(https://lh3.googleusercontent.com/4CefoTb07DPKeJgwA8neHS1bWCV13lCqXZd52jQ-hnggf2vQYEU_JMcj12TaJZr5kqXMKznM55L0fR43=s300);"  data-orig-width="1000" data-orig-height="697" >
 		<!-- VIDEO TEMPLATE -->
@@ Line 464: / Line 554: @@
-<br class="upper-line-break" />
+<div class="element-placeholder" data-elementtype='TITLE' style="display:none;"></div>
-<div class="preview-title-holder removable-parent order-handle">
-		<a href="https://xprs.imcreator.com/viewer/vbid-59a5b-fnxcttvm/vbid-5f09d343-lok1anrm"  data-link-type="EXISTING" target="_self" >
+<div class="element-placeholder" data-elementtype='DIVIDER' style="display:none;"></div>
-	<h2 id="vbid-a18d59f0-6wai9v3d" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >June</h2>
-	</a>
-</div>
-<br class="lower-line-break" />
@@ Line 479: / Line 565: @@
 <div class="element-placeholder" data-elementtype='SUBTITLE' style="display:none;"></div>
-<div class="element-placeholder" data-elementtype='DIVIDER' style="display:none;"></div>
@@ Line 500: / Line 582: @@
 											<div class="preview-item-links order-handle ">
 	<div class="preview-links-wrapper">
+		<a class="removable-parent" href="https://2017.igem.org/Team:Cadets2Vets/Notebook_June" data-link-type="EXTERNAL"   target="_blank" >
+		<span id="vbid-0e6009b3-ss9lvwzx"  class="preview-element Link item-link magic-circle-holder text-element " data-menu-name="PREVIEW_LINK" >June Reports</span>
+			</a>
 <div class="element-placeholder" data-elementtype='LINK' style="display:none;"></div>
@@ Line 534: / Line 622: @@
-		<div  id="vbid-a18d59f0-21vrt6ie"  class="sub item-box  page-box style-a18d59f0-9hy0h3os          " data-holder-type="page"" data-holder-type="page"  data-child-type="STYLE"  data-styleid="style-a18d59f0-9hy0h3os" data-preview-styleid='style-a18d59f0-9hy0h3os' data-preset-type-id="UNRESOLVED">
+		<div  id="vbid-0e6009b3-d6tjyrar"  class="sub item-box  page-box style-0e6009b3-uz8lja45          " data-holder-type="page"" data-holder-type="page"  data-child-type="STYLE"  data-styleid="style-0e6009b3-uz8lja45" data-preview-styleid='style-0e6009b3-uz8lja45' data-preset-type-id="UNRESOLVED">
 			<div class="page-wrapper item-wrapper ">
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+	<a class="image-link top-layer not-wrapping" href="https://2017.igem.org/Team:Cadets2Vets/Notebook_July"  data-link-type="EXTERNAL" target="_blank" ></a>
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+		<a class="removable-parent" href="https://2017.igem.org/Team:Cadets2Vets/Notebook_July" data-link-type="EXTERNAL"   target="_blank" >
+		<span id="vbid-0e6009b3-8tzwtmxr"  class="preview-element Link item-link magic-circle-holder text-element " data-menu-name="PREVIEW_LINK" >July Reports</span>
+			</a>
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+	<a class="image-link top-layer not-wrapping" href="https://2017.igem.org/Team:Cadets2Vets/Notebook_August"  data-link-type="EXTERNAL" target="_blank" ></a>
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 											<div class="preview-item-links order-handle ">
 	<div class="preview-links-wrapper">
+		<a class="removable-parent" href="https://2017.igem.org/Team:Cadets2Vets/Notebook_August" data-link-type="EXTERNAL"   target="_blank" >
+		<span id="vbid-0e6009b3-w0xquhbj"  class="preview-element Link item-link magic-circle-holder text-element " data-menu-name="PREVIEW_LINK" >August Reports</span>
+			</a>
 <div class="element-placeholder" data-elementtype='LINK' style="display:none;"></div>
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+		<div  id="vbid-a7e882ca-zsa07wpt"  class="master item-box  gallery-box style-a7e882ca-8xhe7y59          " data-holder-type="gallery"" data-holder-type="gallery"  data-child-type="STYLE"  data-styleid="style-a7e882ca-8xhe7y59" data-preview-styleid='style-a7e882ca-8xhe7y59' data-preset-type-id="TEXT_BLOCK">
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 			<div class="page-wrapper item-wrapper ">
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-			<div class="helper-div middle-left" >
+			<div class="helper-div middle-center" >
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+	<h2 id="vbid-e1119fa1-sgdaai1b" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >INTER-TEAM MEETING<br></h2>
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-		<p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="height: auto; width: auto; position: relative; font-size: 10px;">Experiments Performed:</span></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="height: auto; width: auto; position: relative; font-size: 10px;"></span><span style="height: auto; width: auto; position: relative; font-size: 10px;">Testing a circuit for biological competency within transformed E.coli.</span></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><br></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="height: auto; width: auto; position: relative; font-size: 10px;">Why We Performed them:</span></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><br></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="height: auto; width: auto; position: relative; font-size: 10px;">These experiments were performed to test for the presents of GFP within E. coli cells after exposure to Arsenate and arsenite.</span></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><br></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><br></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="height: auto; width: auto; position: relative; font-size: 10px;">Results: </span></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="height: auto; width: auto; position: relative; font-size: 10px;">The resulting cultures were inconclusive while consistently negative (no       growth) at the 500uM concentration across all three types. The remaining cultures after the initial concentration were 50/50 in terms of E. coli growth. When checked for fluorescence all samples below 100uM showed possible signal from GFP. However further analysis showed an odd mitigating variable, being that the culture media has an auto-fluorescence within the same spectra as GFP. To prove GFP signal each culture was pelleted out via centrifugation, washed with PBS twice and suspended to eliminate any chance of background signal. The results showed little to no GFP signal. However due to culture variability, this will be replicated. Glycerol stocks have also been created of the stock circuit.</span></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><br></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><br></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><br></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="height: auto; width: auto; position: relative; font-size: 10px;">Moving Forward:</span></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="height: auto; width: auto; position: relative; font-size: 10px;">Due to the results above due to many mitigating factors the cell may not be biologically competent. However, because of the variability between replicates of the varying culture concentrations that will need to be repeated. In parallel to the replication of the culture study, a miniprep and paper based assay will be looked at as well utilizing the Promega S30 kit.</span></p>
+		<p style="line-height: 1.5em; padding-right: 15px; padding-left: 15px; font-size: 13px; color: rgb(0, 0, 0); letter-spacing: 0.6px;">In an attempt to learn the methods used by other IGEM teams we participated in an inter-team Skype call. We chose to meet with the other teams sponsored by the United States Armed forces because we share many of the same challenges. Our notes were as follows:</p><ul style="line-height: 1.5em; margin: 0.3em 0px 0px 1.6em; font-size: 12.8px; font-family: arial, sans-serif; letter-spacing: 0.6px; color: rgb(34, 34, 34);"><li style="margin-left: 15px;"><span style="font-size: 11px;"><span style="font-family: arial;">Every member attending the Jamboree needs to be able to describe an answer questions about the entire project. It could possibly be a handicap to send people who aren't fully on board with the project.</span></span></li><li style="margin-left: 15px;"><span style="font-size: 11px;"><span style="font-family: arial;">One of the Navy programmers mentioned some sort of a universal page that affects all of our team pages. Josh, maybe you can look for that and apply some of our sidebar stuff there.</span></span></li><li style="margin-left: 15px;"><span style="font-size: 11px;"><span style="font-family: arial;">For our presentation, we should go as broad as we can without being boring and then get specific when judges and others ask.</span></span></li><li style="margin-left: 15px;"><span style="font-size: 11px;"><span style="font-family: arial;">The judges really like lots of pictures and a pretty layout for the poster and powerpoint presentation.</span></span></li><li style="margin-left: 15px;"><span style="font-size: 11px;"><span style="font-family: arial;">We still need to fill out our InterLab Excel files and go through the protocols.</span></span></li><li style="margin-left: 15px;"><span style="font-size: 11px;"><span style="font-family: arial;">We need to submit parts. I didn't get the chance to ask how we can check if a part is working, but both teams said we could feel free to email them if we had questions. The Air Force team email is <a target="_blank" style="text-decoration-line: underline; text-decoration-color: rgb(51, 153, 255); color: rgb(17, 85, 204); background: url(&quot;https://2017.igem.org/wiki/skins/Igem/resources/mail_icon.gif&quot;) right center no-repeat; font-weight: bold; transition: all 0.4s ease; padding-right: 18px;" data-cke-saved-href="mailto:igem@carrollhs.org" href="mailto:igem@carrollhs.org">igem@carrollhs.org</a>.</span></span></li><li style="margin-left: 15px;"><span style="font-size: 11px;"><span style="font-family: arial;">The judges use a VGA cord to attach to computers (one of the bigger ones that needs to be screwed in). No internet is provided, but we could use hotspot if needed.</span></span></li><li style="margin-left: 15px;"><span style="font-size: 11px;"><span style="font-family: arial;">Conceptual models make good pictures, even if we can only model a piece of our project. There are good poster examples on iGEM.</span></span></li><li style="margin-left: 15px;"><span style="font-size: 11px;"><span style="font-family: arial;">For our procedures on our presentation and poster, we should include project-specific protocols (e.g. the lyophilization process or testing for GFP) before anything more basic like PCR, but it might be good to specify our specific reagents and primers used for more common things.</span></span></li><li style="margin-left: 15px;"><span style="font-size: 11px;"><span style="font-family: arial;">A lot of projects don't end up working, but some teams cheat and do a project for multiple years in order to make it work, or they stack a team with a ridiculous number of people. It seems judges might (?) be a little bit lenient with those who fudge the rules.</span></span></li><li style="margin-left: 15px;"><span style="font-size: 11px;"><span style="font-family: arial;">The Carroll College team has already gone through the InterLab procedures.</span></span></li><li style="margin-left: 15px;"><span style="font-size: 11px;"><span style="font-family: arial;">We need to specify our part for the GFP we're using.</span></span></li><li style="margin-left: 15px;"><span style="font-size: 11px;"><span style="font-family: arial;">For best human practices integration, a lot of teams go all out and serve food or draw pictures with GFP, so we need to do similar if we want to be competitive. Could we incorporate the Science Breakfasts somehow? We could also practice talking about our project to people at these events.</span></span></li></ul>
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+		<p><span style="color: rgb(0, 0, 0); letter-spacing: 0.6px; background-color: rgb(97, 82, 39);">With the help of high school interns Keshava Katti, Kian Croston, and Josh Grace, senior technician Ryan Brisban performed multiple quantitative and qualitative protein assays to assess the presence of GFP in situ. To these ends, 8 circuit samples were put in a Light Cycler to incubate for 8 hours at 37 degrees Celsius. 4 samples were run with no Arsenic regulator present, meaning that GFP should have been produced continuously for the entire 8 hour period. 3 were run as controls with only broth and no circuit, and 1 control of pure molecular water was run as a baseline measurement. After 8 hours, the normalized fluorescence of the 4 samples containing GFP was significantly higher than any of the controls. To supplement these findings, the Cadets2Vets team ran an acrylamide protein gel on the samples, and although the gel broke in processing, there appeared to be a protein band corresponding to GFP’s known molecular size in kilo-Daltons. A second acrylamide protein gel assay is currently being run to increase the number of trials and provide better evidence than the broken gel. A second assay was also run using Qubit’s total protein assay. All samples were spec’d after creating a linear model of RFUs using standards, but they exceeded the range of the sensor, and thus needed to be diluted. After dilution, the protein concentration was determined to be in the ~1000s of ng/µL for each of the seven samples containing broth, whereas the sample containing molecular water exhibited control values too low for the Qubit to read. The 4 samples containing no Arsenic regulator with the circuit appeared to have lower protein concentrations by ~100-200 ng/ µL than the 3 samples containing only reagents and water. This finding was unexpected to the Cadets2Vets team, who hope that further analysis and measurement will determine the source of the extra protein. The current hypothesis is that the samples containing our circuit functionalized more of the reactants from the reaction matrix.</span></p>
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 <div class="preview-body-holder removable-parent order-handle">
-	<div id="vbid-d499ca04-ltotjw0e" class="preview-element preview-body magic-circle-holder text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_BODY">
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-		<p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="font-size:10px;"><span style="height: auto; width: auto; position: relative;">Experiments Performed:</span></span></p><p style="line-height: 1.5em; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="font-size:10px;">     Validate the use of LC 480 II utilizing T7 constitutive GFP construct.</span></p><p style="line-height: 1.5em; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><br></p><p style="line-height: 1.5em; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="font-size:10px;">     QC/ QA of Ars. 1.1 plasmid cel</span></p><p style="line-height: 1.5em; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><br></p><p style="line-height: 1.5em; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="font-size:10px;">     Culture of Ars 1.1 transformed E. coli were exposed to 25 uM Aresnate +          Aresnite at 37 C in the LC 480 II for 24 hours</span></p><p style="line-height: 1.5em; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><br></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="font-size:10px;"><span style="height: auto; width: auto; position: relative;">Why We Performed them:</span></span></p><p style="line-height: 1.5em; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="font-size:10px;">    We need a way to properly quantify GFP production and QC/QA the                   plasmid</span></p><p style="line-height: 1.5em; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><br></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="font-size:10px;"><span style="height: auto; width: auto; position: relative;">Results:</span></span></p><p style="line-height: 1.5em; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="font-size:10px;">     Validating the use of LC 480 II utilizing T7 constitutive GFP construct</span></p><p style="line-height: 1.5em; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="font-size:10px;">         The LC 480 II was successfully able to monitor the increase GFP over the          incubation time</span></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="font-size:10px;">    QC/QA the plasmid</span></p><p><span style="font-size:10px;">           Circularized plasmid gels proved inconclusive however M13 amplification                did provide strong indication of proper  fragment. The fragment size is 1461            bp, with the second bright band in the ladder being equal to 1650 bp. All                minipreps exhibit a band slight below the 1625 bp band of the ladder,                      which is evident of proper fragment size.</span></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="font-size:10px;"><span style="height: auto; width: auto; position: relative;">Moving Forward:</span></span><br></p><p style="line-height: 1.5em; font-size: 13px; font-family: Verdana, Arial, Helvetica, sans-serif; overflow: visible;"><span style="font-size:10px;">     TBD</span></p>
+		<p><span style="color: rgb(0, 0, 0); letter-spacing: 0.6px; background-color: rgb(97, 82, 39);">On July 19, 2017, senior technician Ryan Brisban with the help of high school interns Keshava Katti, Kian Croston, and Josh Grace analyzed a second acrylamide protein assay. This was done in order to qualitatively demonstrate the presence of GFP in situ for Cadets2Vets’ iGEM arsenic-sensing circuit, and increase the power of the evidence collected from the previous acrylamide protein assay whose gel broke. The second acrylamide protein gel assay presents strong qualitative evidence for the presence of GFP and known regulatory proteins in situ. The fact that the Cadets2Vets team’s regulated plasmids appear to have a high degree of variability (Fig 1.1) and protein smearing leads the team to believe that a shielding mechanism is occurring where the excess regulatory protein in situ prevents GFP from presenting as strongly as it otherwise could. The Cadets2Vets team plans to next perform ticketing and brainstorm methods to increase GFP signaling efficiency.</span></p>
 	</div>
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+<br class="upper-line-break" />
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+		<p><em style="color: rgb(0, 0, 0); font-size: 11px; letter-spacing: 0.6px; text-align: center;">Fig 1.1 – Acrylamide protein gel assay. First ladder (far left) is a standard. Second, third, and fourth are reaction replicates of an unregulated GFP promoter complex, with two strongly visible bands (bottom of latter bands demonstrate presence of reaction proteins at 13.3 kilo-Daltons; top of ladder bands demonstrate presence of GFP at 32.7 kilo-Daltons). The fifth ladder is a reaction control with no plasmid (bottom band presenting for reaction proteins). The sixth, seventh, and eighth ladders contain reaction replicates of the Cadets2Vets team’s regulated GFP plasmid. Variability demonstrated is due to varying amounts of regulatory protein and variability.</em></p>
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 <div class="preview-title-holder removable-parent order-handle">
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+	<h2 id="vbid-2f7b59fe-fyvid0oq" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >July 20, 2017<br></h2>
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+		<p class="c6" style="color: rgb(0, 0, 0); font-size: 11pt; font-family: Arial; padding-top: 0pt; padding-bottom: 10pt; line-height: 1.15; letter-spacing: normal;"><span class="c0" style="font-size: 12pt; font-family: Calibri;">On July 20, 2017, senior technician Ryan Brisban with the help of high school interns Keshava Katti, Kian Croston, and Josh Grace created tickets by aliquoting </span><span class="c0 c8" style="font-size: 12pt; font-family: Calibri; font-style: italic;">Ars 1.1</span><span class="c1 c0" style="vertical-align: baseline; font-size: 12pt; font-family: Calibri;"> circuits into wells with various other reagents. This was done to test the in-paper efficacy of the circuits that the Cadets2Vets team engineered.</span></p><p class="c6" style="color: rgb(0, 0, 0); font-size: 11pt; font-family: Arial; padding-top: 0pt; padding-bottom: 10pt; line-height: 1.15; letter-spacing: normal;"><span class="c1 c0" style="vertical-align: baseline; font-size: 12pt; font-family: Calibri;">        To these ends, the Cadets2Vets team aliquoted their circuit and reaction solution without arsenic ions to each well of the negative control. The Cadets2Vets team aliquoted their circuit and reaction solution with arsenic ions into each well of the positive control.</span></p><p class="c6" style="color: rgb(0, 0, 0); font-size: 11pt; font-family: Arial; padding-top: 0pt; padding-bottom: 10pt; line-height: 1.15; letter-spacing: normal;"><span class="c0" style="font-size: 12pt; font-family: Calibri;">        The reaction solution contained 7.5 </span><img data-cke-saved-src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL" src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL"><span class="c0" style="font-size: 12pt; font-family: Calibri;">S30 Extract, 10 </span><img data-cke-saved-src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL" src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL"><span class="c0" style="font-size: 12pt; font-family: Calibri;">S30 Promega, 2.5 </span><img data-cke-saved-src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL" src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL"><span class="c0" style="font-size: 12pt; font-family: Calibri;">amino (+), 0.5 </span><img data-cke-saved-src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL" src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL"><span class="c0" style="font-size: 12pt; font-family: Calibri;">amino (-), and 2 </span><img data-cke-saved-src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL" src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL"><span class="c0" style="font-size: 12pt; font-family: Calibri;">molecular water. For the positive test, 2.5 </span><img data-cke-saved-src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL" src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL"><span class="c0" style="font-size: 12pt; font-family: Calibri;">of our circuit plasmid and machinery was added, and the 2 </span><img data-cke-saved-src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL" src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL"><span class="c0" style="font-size: 12pt; font-family: Calibri;">molecular water contained arsenite and arsenate at a 150 </span><img data-cke-saved-src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DM" src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DM"><span class="c0" style="font-size: 12pt; font-family: Calibri;"> concentration. 2 </span><img data-cke-saved-src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL" src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL"><span class="c0" style="font-size: 12pt; font-family: Calibri;">of water (molecular for negative control, arsenic ion and water mixture for positive test) was added to wells in column 2, and 4 </span><img data-cke-saved-src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL" src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DL"><span class="c0" style="font-size: 12pt; font-family: Calibri;">of water (molecular for negative control, arsenic ion and water mixture for positive test) was added to wells in column 3. The wells in the second column had an arsenic ion concentration of 82.5 </span><img data-cke-saved-src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DM" src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DM"><span class="c0" style="font-size: 12pt; font-family: Calibri;">and the wells in the third column has an arsenic ion concentration of 208.3 </span><img data-cke-saved-src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DM" src="https://www.google.com/chart?cht=tx&amp;chf=bg,s,FFFFFF00&amp;chco=000000&amp;chl=%5Cmu%7B%7DM"><span class="c1 c0" style="vertical-align: baseline; font-size: 12pt; font-family: Calibri;">for the positive test ticket.</span></p><p class="c4" style="color: rgb(0, 0, 0); font-size: 11pt; font-family: Arial; padding-top: 0pt; text-indent: 36pt; padding-bottom: 10pt; line-height: 1.15; letter-spacing: normal;"><span class="c1 c0" style="vertical-align: baseline; font-size: 12pt; font-family: Calibri;">These tickets were then let to incubate overnight (~18 hours) at ~40 degrees Celsius, at which point they were analyzed qualitatively using a backlight (Fig 1.1). Although measures were taken to try to prevent the tickets from drying up by supplying a source of humidity in the petri dishes, the Negative Control Paper Circuit unfortunately dried out slightly, which is why some of the wells seem lighter than the others. Despite this, no fluorescence was detected on any of the negative control wells. The positive test exhibited fluorescence in the first column of wells, but the second and third wells exhibited no fluorescence. The Cadets2Vets team currently hypothesizes that this may be a result of arsenic ions saturating the pores of the ticket, inhibiting the function of the cellular machinery by destabilizing GFP conformation, or negatively influencing ligand effects, preventing the production of GFP.</span></p><p class="c4" style="color: rgb(0, 0, 0); font-size: 11pt; font-family: Arial; padding-top: 0pt; text-indent: 36pt; padding-bottom: 10pt; line-height: 1.15; letter-spacing: normal;"><span class="c0" style="font-size: 12pt; font-family: Calibri;">Moving forward, the Cadets2Vets team plans to amplify the signal of the positive test by spiking in more regulatory circuits to the abiotic wells. The team hypothesizes that this allow more of the arsenic ions to be functionalized by the regulatory proteins to produce GFP, increasing the ratio of GFP created. The Cadets2Vets team also plans to alter the percentage-based concentrations of arsenic in the positive test wells to determine the current upper abiotic LOD. Lastly, the Cadets2Vets team will test new methods to keep the tickets from drying out during incubation to find a (hopefully) more effective method than what is currently being used.</span></p>
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+		<p><em><span style="color:#000000;"><span style="font-family: Raleway; letter-spacing: normal;">Fig 1.1 – Fluorescence seen on positive test ticket (right), with no fluorescence on negative control ticket. Discoloration is due to drying of ticket and biological variability.</span></span></em></p>
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+		<p dir="ltr" style="line-height:1.3800000000000001;margin-top:0pt;margin-bottom:10pt;"><span id="docs-internal-guid-95d902ce-9ae6-86ee-9e30-8a2338706c57"><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">    </span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">On July 25, 2017, senior technician Ryan Brisban with the help of high school interns Keshava Katti, Kian Croston, and Josh Grace ran a second ticket assay to increase the power of the previous test and provide clearer results.</span></span></p><p dir="ltr" style="line-height:1.3800000000000001;margin-top:0pt;margin-bottom:10pt;"><span id="docs-internal-guid-95d902ce-9ae6-86ee-9e30-8a2338706c57"><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">    </span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">To these ends, the Cadets2Vets team aliquoted their circuit and reaction solution without arsenic ions to each well of the negative control. The Cadets2Vets team aliquoted their circuit and reaction solution with arsenic ions into each well of the positive control.</span></span></p><p><span id="docs-internal-guid-95d902ce-9ae6-86ee-9e30-8a2338706c57"><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">    </span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">The reaction solution contained 7.5 </span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">S30 Extract, 10 </span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">S30 Promega, 2.5 </span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">amino (+), 0.5 </span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">amino (-), and 2 </span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">molecular water. For the positive test, 2.5 </span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">of our circuit plasmid and machinery was added, and the 2 </span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">molecular water contained arsenite and arsenate at a 150 </span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">M</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"> concentration. The negative control ticket used only 2 </span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">of reaction mixture for the first two columns of wells on the left, and the other two columns of wells were filled with 2 </span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">of molecular water. The positive test ticket used 2 </span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">of the test mixture in the first column of wells, and 4</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">of test mixture in the second column of wells. The other two columns of wells were filled with 2</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">of molecular water.<strong></strong></span></span><span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"></span></span><br></p><p><span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"></span></span><br></p><p dir="ltr" style="line-height:1.3800000000000001;margin-top:0pt;margin-bottom:10pt;text-indent: 36pt;"><span id="docs-internal-guid-95d902ce-9ae6-d923-ed6e-ba3d17140623"><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">These tickets were then let to incubate overnight (~18 hours) at ~40 degrees Celsius, at which point they were analyzed qualitatively using a backlight (Fig 1.1 and Fig 1.2). Once again, no fluorescence was detected on any of the negative control wells. The positive test exhibited fluorescence in the first and second columns of wells, and qualitatively it appears that there may have been some form of dose response, as the second column of wells appears brighter. The negative control appears to have dried again slightly, and the Cadets2Vets team will be aliquoting more reaction mixture into each well during later tests to try and combat this issue.</span></span></p><div><br></div><span><span style="font-size: 12pt; font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"><strong></strong></span></span>
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+					<div id="no-image-holder"  class="preview-image-holder inner-pic-holder" data-menu-name="PREVIEW_INLINE_IMAGE_HOLDER">
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@@ Line 1,571: / Line 2,641: @@
 <br class="upper-line-break" />
-<div class="preview-title-holder removable-parent order-handle">
+<div class="preview-body-holder removable-parent order-handle">
-	<h2 id="vbid-8bb6e0b7-zc2jpxkd" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >This is a M13 confirmation fragment insert. of arsenic circuit. Top band is confirmation of presence of insert at approximately 1.4 kb<br></h2>
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+		<p><span style="font-size:10px;"><em><span id="docs-internal-guid-95d902ce-9ae8-34c7-0eb0-fc1cd115889e"><span style="font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">Fig 1.1 and Fig 1.2 - The Positive Test Circuit (left) with first row containing 2 </span><span style="font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">of test mixture, and the second row containing 4 </span><span style="font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">of test mixture. Possible dose response is visible. The Negative Control Circuit (right), containing 2 </span><span style="font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">L</span><span style="font-family: Calibri; color: rgb(0, 0, 0); background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"> of reaction mixture in first and second columns.</span></span></em></span></p>
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+				<a class='social-link-url' href="https://www.youtube.com/channel/UCuVc4cKxuUzpjZfywBgoT9w" target='_blank'><img class='preview-link-img ' src="//xprs.imcreator.com/images/socialmedia/7youtube.png" ></a>
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+				<a class='social-link-url' href="https://plus.google.com/u/0/+RainincubatorOrg" target='_blank'><img class='preview-link-img ' src="//xprs.imcreator.com/images/socialmedia/7googleplus.png" ></a>
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