Team:Brasil-SP/Notebook

From 2014.igem.org

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<a href="https://static.igem.org/mediawiki/2014/e/eb/Assembly_Form.pdf"><img src="https://static.igem.org/mediawiki/2014/3/32/Assembly_form_pg3.png" style="width:700px;height:400px"/></a>
<a href="https://static.igem.org/mediawiki/2014/e/eb/Assembly_Form.pdf"><img src="https://static.igem.org/mediawiki/2014/3/32/Assembly_form_pg3.png" style="width:700px;height:400px"/></a>
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      <a href="https://static.igem.org/mediawiki/2014/3/38/AII.pdf"><img src="https://static.igem.org/mediawiki/2014/e/e1/AII_image.png" width="100%" height="150px"/></a>
 
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      <a href="https://static.igem.org/mediawiki/2014/d/d6/AIII.pdf"><img src="https://static.igem.org/mediawiki/2014/a/ad/AIII_image.png" width="100%" height="150px"/></a>
 
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      <a href="https://static.igem.org/mediawiki/2014/e/eb/AIV.pdf"><img src="https://static.igem.org/mediawiki/2014/4/49/AIV_image.png" width="100%" height="150px"/></a>
 
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      <a href="https://static.igem.org/mediawiki/2014/e/ea/BIII.pdf"><img src="https://static.igem.org/mediawiki/2014/3/36/BIII.png"width="100%" height="150px"/></a>
 
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<h1 align=center>Characterization Assemblies</h1>
 
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<p> Apart from the main genetic circuit we also assembled others for characterization purposes, such as the validation of the promoters and tunning of our threshold setter concentration, the QteE.</p>
 
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<h2>Promoter BBa_K823003</h2>
 
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<p><strong>Question</strong>: Does the constitutive promoter BBa_K823003 work properly?</p>
 
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<img src="https://static.igem.org/mediawiki/2014/d/d5/KI_w.png" width="300" height="170px"/>
 
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<p><strong>Results</strong>: After the incubation period of the transformed <em>E. coli</em> a large portion of the colonies were glowing green. So the promoter does work. Moreover, this biobrick works on <em>E. coli</em> despite the fact it was designed for <em>B. subtilis</em>.</p>
 
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<img src="https://static.igem.org/mediawiki/2014/e/ef/Glowing_E_coli.JPG" width="400px" height="200px"/>
 
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<h2>Promoter BBa_K143015</h2>
 
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<p><strong>Question</strong>: How does the transcription caused by this promoter varies with the IPTG induction?</p>
 
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<img src="https://static.igem.org/mediawiki/2014/7/7a/KXVI_w.png" width="600px" height="350px"/>
 
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<p><strong>Results</strong>:</p>
 
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<h2>Tunning of the QteE Threshold</h2>
 
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<p><strong>Question</strong>:What are the concentration of QteE needed to hamper the LasR induction of the promoter PlasR?</p>
 
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<p>This is the most difficult task of our project. Tunning the production of QteE so that we establish the correct threshold for the discretization of the Cystatin C level in serum. To attack this challenge we designed 3 circuits so that we could plot a calibration curve. In this circuits we put the transcription of the LasR and QteE under two differnt promoters, the Pveg (BBa_K823003) and PlasR (BBa_K143015).</p>
 
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<img src="https://static.igem.org/mediawiki/2014/8/86/KXV_w.png" width="600px" height="300px"/>
 
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<img src="https://static.igem.org/mediawiki/2014/c/c4/KXIII_w.png" width="600px" height="300px"/>
 
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<h1>Assembly Forms<h1>
 
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      <a href="https://static.igem.org/mediawiki/2014/9/90/KI.pdf"><img src="https://static.igem.org/mediawiki/2014/2/23/KI.png"width="100%" height="150px"/></a>
 
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      <a href="https://static.igem.org/mediawiki/2014/b/b3/KII.pdf"><img src="https://static.igem.org/mediawiki/2014/c/c5/KII.png"width="100%" height="150px"/></a>
 
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      <a href="https://static.igem.org/mediawiki/2014/7/78/KIII.pdf"><img src="https://static.igem.org/mediawiki/2014/e/ed/KIII.png"width="100%" height="150px"/></a>
 
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      <a href="https://static.igem.org/mediawiki/2014/9/95/KIV.pdf"><img src="https://static.igem.org/mediawiki/2014/e/ee/KIV.png"width="100%" height="150px"/></a>
 
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      <a href="https://static.igem.org/mediawiki/2014/3/3d/KV.pdf"><img src="https://static.igem.org/mediawiki/2014/a/ac/KV.png"width="100%" height="150px"/></a>
 
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      <a href="https://static.igem.org/mediawiki/2014/8/83/KVI.pdf"><img src="https://static.igem.org/mediawiki/2014/3/37/KVI.png"width="100%" height="150px"/></a>
 
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      <a href="https://static.igem.org/mediawiki/2014/9/95/KX.pdf"><img src="https://static.igem.org/mediawiki/2014/d/d5/KX.png"width="100%" height="150px"/></a>
 
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      <a href="https://static.igem.org/mediawiki/2014/9/9c/KXI.pdf"><img src="https://static.igem.org/mediawiki/2014/b/b8/KXI.png"width="100%" height="150px"/></a>
 
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      <a href="https://static.igem.org/mediawiki/2014/6/67/KXIV.pdf"><img src="https://static.igem.org/mediawiki/2014/c/c5/KXIV.png"width="100%" height="150px"/></a>
 
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      <li><a href="https://static.igem.org/mediawiki/2014/b/b7/AI.pdf">AI</a></li>
 
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<h1 align=center><strong>Life Inside the LAB</strong></h1>
<h1 align=center><strong>Life Inside the LAB</strong></h1>
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     <td>30/06
     <td>30/06
     <ul>
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       <li><a href="https://static.igem.org/mediawiki/2014/7/74/PCR.pdf" style="color:#f05151">PCR</a> lasR (BBa_C0079). The purpose of this PCR was to add the RBS (BBa_K143021), through addition of the sequence in the primer foward, and also to remove the LVA tag. In our primers we only added the restriction sites X and S, so we still need to put it in the pSB1C3 for the Biobrick standard completion. When standardized this part will be called BBa_K1521001.</li>
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       <li><a href="https://static.igem.org/mediawiki/2014/7/74/PCR.pdf" style="color:#f05151">PCR</a> lasR (<a href="http://parts.igem.org/Part:BBa_C0079" style="color:#3ab473">BBa_C0079</a>). The purpose of this PCR was to add the RBS (<a href="http://parts.igem.org/Part:BBa_K143021" style="color:#3ab473">BBa_K143021</a>), through addition of the sequence in the primer foward, and also to remove the LVA tag. In our primers we only added the restriction sites X and S, so we still need to put it in the pSB1C3 for the Biobrick standard completion. When standardized this part will be called BBa_K1521001.</li>
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         <li>BBa_B0015 (restriction analysis fail, repeat)</li>
         <li>BBa_B0015 (restriction analysis fail, repeat)</li>
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       <li><a href="https://static.igem.org/mediawiki/2014/7/74/PCR.pdf" style="color:#f05151">PCR</a> the qteE gene for amplification. We also added the RBS (BBa_K143021) using the foward primer. In our primers we only added the restriction sites X and S, so we still need to put it in the pSB1C3 for the Biobrick standard completion. When standardized this biobrick will be called BBa_K1521000.</li>
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       <li><a href="https://static.igem.org/mediawiki/2014/7/74/PCR.pdf" style="color:#f05151">PCR</a> the qteE gene for amplification. We also added the RBS (<a href="http://parts.igem.org/Part:BBa_K143021" style="color:#3ab473">BBa_K143021,</a>) using the foward primer. In our primers we only added the restriction sites X and S, so we still need to put it in the pSB1C3 for the Biobrick standard completion. When standardized this biobrick will be called BBa_K1521000.</li>
       <li><a href="https://static.igem.org/mediawiki/2014/0/03/PCR_Product_Clonig.pdf" style="color:#f05151">Ligation</a> of RBS+lasR in PTZ57R/T vector (instaclone kit)</li>
       <li><a href="https://static.igem.org/mediawiki/2014/0/03/PCR_Product_Clonig.pdf" style="color:#f05151">Ligation</a> of RBS+lasR in PTZ57R/T vector (instaclone kit)</li>
     </ul>
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Revision as of 06:04, 16 October 2014


Main Assembly Map

AI AII AIII BIII AIV BII CII DI AVI AV

Assemblies forms

Assembly form template

This is the template designed to help with the laboratory organization during the assemble of biological parts. If you want to use the assembly method we used you can print the form and complete it for each construction. Hope it helps :)

Life Inside the LAB

July

Monday Tuesday Wednesday Thursday Friday Saturday Sunday
30/06
  • PCR lasR (BBa_C0079). The purpose of this PCR was to add the RBS (BBa_K143021), through addition of the sequence in the primer foward, and also to remove the LVA tag. In our primers we only added the restriction sites X and S, so we still need to put it in the pSB1C3 for the Biobrick standard completion. When standardized this part will be called BBa_K1521001.
 01/07 02/07
  • Transformation of the Biobricks:
    • BBa_143012
    • BBa_K081001
    • BBa_E0840
  • Inoculum of the Biobricks sent by iGEM HQ:
    • BBa_K316037
    • BBa_K316018
    • BBa_K316015
 03/07 04/07 05/07 06/07
  • Inoculum:
    • BBa_143012
    • BBa_K081001
    • BBa_E0840
07/07 08/07 09/07
  • Inoculum:
    • BBa_B0015
 10/07
  • Miniprep, Quantification and Restriction Analysis:
    • BBa_B0015 (restriction analysis fail, repeat)
  • PCR the qteE gene for amplification. We also added the RBS (BBa_K143021,) using the foward primer. In our primers we only added the restriction sites X and S, so we still need to put it in the pSB1C3 for the Biobrick standard completion. When standardized this biobrick will be called BBa_K1521000.
  • Ligation of RBS+lasR in PTZ57R/T vector (instaclone kit)
 11/07 12/07 13/07
14/07 15/07 16/07
  • Transformation:
    • BBa_K823003
    • RBS+qteE
  • Electrophoresis analysis:
    • BBa_B0015
 17/07 18/07 19/07 20/07
21/07 22/07 23/07 24/07 25/07 26/07 27/07
28/07
  • Assembly AIII:
    • Restriction analysis (+NdeI enzyme)
    • OBS: This analysis was reapeted because we had problems confirming the assembly. The issue was that both the construction and the pSB1C3 vector had similar size.
  • Assembly AII, AV and AVI:
 29/07 30/07 31/07
  • Assembly AII, AV and AVI:
    • Inoculum
  • PCR BBa_K143055 for RBS removal
  • OBS: This was one of the parts sent by the Imperial College Team. Because of a comunication failure we assumed that this part was comming without the RBS, so we included a RBS in the parts we were going to use with this Lac promotor :)
  • PCR of Plac BBa_K143055 was followed by a Gel Purification and Ligation in the PUC19 vector



August

Monday Tuesday Wednesday Thursday Friday Saturday Sunday
01/08 02/08 03/08
04/08 05//08 06/08 07/08 08/08 09/08 10/08
11/08
  • Prepare Plac BBa_K143055 for sequencing
  • Analyse the RBS+qteE and RBS+lasR sequencing file
 12/08
  • Digestion:
    • RBS+qteE
    • RBS+lasR
    • OBS: this digestion was performed so we clone this parts in the pSB1C3 vector to put them in Biobrick standard
 13/08 14/08
  • Gel Purification and Ligation in pSB1C3 for BBiobrick standard:
    • RBS+qteE
    • RBS+lasR
  • Assemblies BII and BIII:
    • Gel Purification
    • OBS: we had some problems here with the purification and we lost all our digestions, so we're repeating the digestion
 15/08 16/08 17/08
18/08 19/08 20/08
  • RBS+qteE, RBS+lasR and Assemblies BII and BIII:
    • Inoculum
 21/08 22/08 23/08 24/08
25/08
  • Inoculum
    • BBa_J04450
    • BBa_K143055
  • PCR BBa_K143055 with higher melting temperature.
 26/08 27/08 28/08 29/08 30/08 31/08



September

Monday Tuesday Wednesday Thursday Friday Saturday Sunday
01/09 02/09 03/09 04/09 05/09 06/09 07/09
08/09 09/09 10/09 11/09 12/09
  • Assemblies DI, KVIII, KX and KXI:
  • We had far to many difficulties trying to remove the RBS from the BBa_K143055, so we decided to synthesize it using PCR.
  • PCR for synthesis of the BBa_143015 (BBa_143055 without RBS)
 13/09
  • Assembly KX
    • Inoculum
  • Assemblies DI, KVIII and KXI:
  • Prepare LB medium (solid and liquid)
 14/09
15/09 16/09 17/09
  • Measurement Interlab Study - Samples growth biobrick devices 1, 2 and 3
 18/09
  • Replate BBa_K143015. We forgot the X-gal :(
  • Measurement Interlab Study - Sample preparation and characterization by Flow Cytometry and Fluorometry
 19/09 20/09 21/09
22/09 23/09 24/09 25/09
  • Assembly KXIV:
    • Inoculum
 26/09 27/09 28/09
29/09 30/09



October

Monday Tuesday Wednesday Thursday Friday Saturday Sunday
01/10 02/10 03/10 04/10 05/10
06/10 07/10 08/10 09/10 10/10 11/10 12/10
13/10 14/10 15/10 16/10 17/10 18/10 19/10
20/10 21/10 22/10 23/10 24/10 25/10 26/10
27/10 28/10 29/10 30/10 31/10

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