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

Based off of the recommendations from previous report, it was determined that the two primary needs were to identify a means of quantifying GFP production, and QC/QA the plasmid.

Proposed route:

Attempt to see if LC 480 II is capable of monitoring GFP production
QC/QA Plasmid:
- Plasmid Gel
- M13 amplification
Attempt to quantify the culture, at 25 µM of arsenite and arsenate

Experiments:

Validate the use of LC 480 II utilizing T7 constitutive GFP construct
- Experiment was run in triplicate with both
  - Controls: T7 expression blank, Solution blank (water), and Plasmid blank
QC/QA of Ars 1.1 plasmid circuit
- Minipreps were done in triplicate and tested on the nanodrop
- The triplicate minipreps were run on three separate gels at 0.7% agarose
- All three mini-preps were tested with an M13 cloning site amplification and the insert size was checked on a 1% agarose gel
Culture of Ars 1.1 transformed E. coli were exposed to 25uM Arsenate+Arsenite at 37C in the LC 480 II for 24 hours
- Percentages are in m/V
  - LB 4%
  - Ampicillin 0.01%

Results:

Validate the use of LC 480 II utilizing T7 constitutive GFP construct- Successful

$C:\Users\Ryan\AppData\Local\Microsoft\Windows\INetCache\Content.Word\IMG_20170628_133008 (1).jpg$

The LC 480 II was successfully able to monitor the increase of GFP over the incubation time.

QC/QA the Plasmid:

Circularized plasmid gels proved inconclusive, however M13 amplification did provide strong indication of the 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 slightly below the 1625 bp band of the ladder, which is evident of the proper fragment size.

$C:\Users\Ryan\AppData\Local\Microsoft\Windows\INetCache\Content.Word\IGEM M13 GEL IN TRIPLICATE 07032017.jpg$

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

Latest revision as of 15:31, 1 November 2017

Our Progress

June Notebook

06/26/2017

IMAGE GALLERY

The lightcycler graphed the normalized fluorescence of various controls.

This is the ticket we ran. The first column is fluorescing, and the other columns are not.

During the initial test of the circuit in the presence of 25 um arsenite and arsenate solution, we were able to generate a fluorescent signal using the Light Cycler.

Follow us on Social Media!

2304 Jefferson Ave, Tacoma, WA 98402

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@@ Line 242: / Line 475: @@
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+		<a class="removable-parent" href="https://2017.igem.org/Team:Cadets2Vets/Notebook_August" data-link-type="EXTERNAL"   target="_blank" >
+		<span id="vbid-2b70f1ab-w0xquhbj"  class="preview-element Link item-link magic-circle-holder text-element " data-menu-name="PREVIEW_LINK" >August Reports</span>
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-	<h2 id="vbid-d74eb2c2-wtwxjk0y" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >Date: June 21, 2017<br></h2>
+	<h1 id="vbid-7a9059f5-etmsamwu" 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 Notebook</h1>
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-	<div id="vbid-d74eb2c2-0uefpxyr" class="preview-element preview-body magic-circle-holder text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_BODY">
-		<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>
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-	<h2 id="vbid-d499ca04-1jkacb2h" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >Date: June 26, 2017<br></h2>
+	<h2 id="vbid-4de3f9f3-1nufibyy" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >06/26/2017</h2>
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-	<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">
+	<div id="vbid-4de3f9f3-yulquncq" class="preview-element preview-body magic-circle-holder text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_BODY">
-		<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 dir="ltr">Notes:</p><p dir="ltr">Based off of the recommendations from previous report, it was determined that the two primary needs were to identify a means of quantifying GFP production, and QC/QA the plasmid.</p><br><p dir="ltr">Proposed route:</p><ul><li dir="ltr"><p dir="ltr">Attempt to see if LC 480 II is capable of monitoring GFP production</p></li><li dir="ltr"><p dir="ltr">QC/QA Plasmid:</p><ul><li dir="ltr"><p dir="ltr">Plasmid Gel</p></li><li dir="ltr"><p dir="ltr">M13 amplification</p></li></ul></li><li dir="ltr"><p dir="ltr">Attempt to quantify the culture, at 25 µM of arsenite and arsenate</p></li></ul><p><br></p><p dir="ltr">Experiments:</p><ul><li dir="ltr"><p dir="ltr">Validate the use of LC 480 II utilizing T7 constitutive GFP construct</p><ul><li dir="ltr"><p dir="ltr">Experiment was run in triplicate with both</p><ul><li dir="ltr"><p dir="ltr">Controls: T7 expression blank, Solution blank (water), and Plasmid blank</p></li></ul></li></ul></li><li dir="ltr"><p dir="ltr">QC/QA of Ars 1.1 plasmid circuit</p><ul><li dir="ltr"><p dir="ltr">Minipreps were done in triplicate and tested on the nanodrop</p></li><li dir="ltr"><p dir="ltr">The triplicate minipreps were run on three separate gels at 0.7% agarose</p></li><li dir="ltr"><p dir="ltr">All three mini-preps were tested with an M13 cloning site amplification and the insert size was checked on a 1% agarose gel</p></li></ul></li><li dir="ltr"><p dir="ltr">Culture of Ars 1.1 transformed E. coli were exposed to 25uM Arsenate+Arsenite at 37C in the LC 480 II for 24 hours</p><ul><li dir="ltr"><p dir="ltr">Percentages are in m/V</p><ul><li dir="ltr"><p dir="ltr">LB 4%</p></li><li dir="ltr"><p dir="ltr">Ampicillin 0.01%</p></li></ul></li></ul></li></ul><p><br><br></p><p dir="ltr">Results:</p><p dir="ltr">Validate the use of LC 480 II utilizing T7 constitutive GFP construct- Successful</p><br><p dir="ltr"><span id="docs-internal-guid-2e42da7c-1809-2e74-a25d-cd92979219b5"><span style="font-size: 11pt; font-family: Calibri; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"><img data-cke-saved-src="https://static.igem.org/mediawiki/2017/2/2c/C2V_Notebook_JuneData1.jpg" src="https://static.igem.org/mediawiki/2017/2/2c/C2V_Notebook_JuneData1.jpg" width="635" height="278" style="border: none; transform: rotate(0.00rad); -webkit-transform: rotate(0.00rad);" alt="C:\Users\Ryan\AppData\Local\Microsoft\Windows\INetCache\Content.Word\IMG_20170628_133008 (1).jpg"></span></span><br></p><p dir="ltr">The LC 480 II was successfully able to monitor the increase of GFP over the incubation time.</p><p><br><br><br></p><p dir="ltr">QC/QA the Plasmid:</p><p dir="ltr">Circularized plasmid gels proved inconclusive, however M13 amplification did provide strong indication of the 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 slightly below the 1625 bp band of the ladder, which is evident of the proper fragment size.</p><p style="text-align: center;"><br><span id="docs-internal-guid-2e42da7c-1809-9bd9-a8ce-7360bc5c67db"><img data-cke-saved-src="https://static.igem.org/mediawiki/2017/6/64/C2V_Notebook_JuneData2.jpg" src="https://static.igem.org/mediawiki/2017/6/64/C2V_Notebook_JuneData2.jpg" width="520" height="605" style="border: none; transform: rotate(0.00rad); -webkit-transform: rotate(0.00rad);" alt="C:\Users\Ryan\AppData\Local\Microsoft\Windows\INetCache\Content.Word\IGEM M13 GEL IN TRIPLICATE 07032017.jpg"></span>
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+	<div  id="vbid-c4fe2e5c-hddajlah" class="inner-pic preview-element  magic-circle-holder  load-high-res " data-menu-name="PREVIEW_INLINE_IMAGE" style="background-image:url(https://static.igem.org/mediawiki/2017/0/05/C2V_Notebook_JuneGalleryMiddle.png);"  data-orig-width="231" data-orig-height="199" >
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@@ Line 1,438: / Line 1,777: @@
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+<div class="element-placeholder" data-elementtype='ICON' style="display:none;"></div>
 <br class="upper-line-break" />
 <div class="preview-title-holder removable-parent order-handle">
-	<h2 id="vbid-9c93986c-wtwxjk0y" 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 the test of T7 constitutiveGFP on a paper based ticket. From left-to-right it is positive control, negative control, water, and another water<br></h2>
+	<h2 id="element-5f8799e2bb5a2dd" 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 the ticket we ran. The first column is fluorescing, and the other columns are not.</h2>
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+	<div  id="vbid-b07c211d-vb18prn7" class="inner-pic preview-element  magic-circle-holder  load-high-res " data-menu-name="PREVIEW_INLINE_IMAGE" style="background-image:url(https://static.igem.org/mediawiki/2017/2/20/C2V_NotebookJuneGalleryRight.jpg);"  data-orig-width="512" data-orig-height="382" >
 		<!-- VIDEO TEMPLATE -->
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 							<div class="preview-content-holder shrinker-content">
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