Team:TU Darmstadt/safety/Labjournal

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<a href="https://2013.igem.org/Team:TU_Darmstadt">
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<img alt="Home_ausgewählt" src="/wiki/images/a/a9/Darmstadt_Taskbar_01_%28Home_angewählt%29.jpg" width="100" height="24"></a>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook">
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<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font>
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<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols">
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<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font>
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<p text-aligne:left style="margin-left:50px; margin-right:50px">
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Assembly of the gBlocks  
+
<b><font size="5" color="#F0F8FF" face="Arial regular">gBlocks assembly</font></b>
<Br><Br>
<Br><Br>
-
Based on the pDawn Plasmid we desinged the light induced kill switch and synthesized the construct as gBlock Fragments on IDT. We assembeled the 10 Fragments follow the Protocol:
 
-
<Br>
+
 
 +
We designed the light induced kill switch based on the pDawn plasmid. We ordered 10 gBlock fragments coding for our plasmid from <a href="http://eu.idtdna.com/"><font size="3" color="#F0F8FF" face="Arial regular"><b>IDT</b></font></a>. We assembeld the fragments according to the following protocol:
 +
 
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<Br><Br>
 +
 
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<img alt="Assembly PCR with 10 gBlocks" src="/wiki/images/0/03/Assembly_pcr.jpg" width="100" height="200" align="right"></a>
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<dl class="igemTUD2013gelpicture" style="margin-right:50px;">
 +
<dd><img alt="Assembly PCR with 10 gBlocks" src="/wiki/images/0/03/Assembly_pcr.jpg" width="100" height="200" align="center"></dd>
 +
<dd><font size="2" color="#F0F8FF" face="Arial regular">Fig. 1:<br>M: GeneRuler DNA Ladder Mix (Thermo Sc.)<br><p>1: Assembly PCR.  The signal at 5 kbps is our construct.</p></font></dd>
 +
</dl>
<ul style="margin-left:50px; margin-right:50px; text-align:justify; ">
<ul style="margin-left:50px; margin-right:50px; text-align:justify; ">
-
<li>Reconstitute the gBlock Fragments in 10 µl TE Buffer</li>
+
<li>Reconstitute the gBlock fragments in 10 µl TE buffer</li>
-
<li>Use 1 µl of each gBlock Fragment for a PCR with the Q5 Polymerase</li>
+
<li>Use 1 µl of each gBlock fragment for a PCR with the Q5 Polymerase</li>
-
<li>Perform the PCR Reaktion with the Primers Prefix and Suffix and an annealing temperature of 55 °C <br> (30 cycles)</li>
+
<li>Perform the PCR reaction with primers coding for the prefix and suffix and an annealing temperature of 55 °C <br>(30 cycles)</li>
-
<li>Take 5 µl of the 50 µl reaction for DNA-gelelectrophoreses</li>
+
<li>Load an 0.8% agarose gel with 5 µl of PCR reaction (total volume 50 µl) and perform a DNA gel electrophoresis</li>
-
<li>Restrict the other 45 µl with the restrictionenzymes EcoRI and PstI (10 U each) for 1 h at 37 °C</li>
+
<li>Cut the other 45 µl with the restriction enzymes EcoRI and PstI (10 U each) for 1 h at 37 °C</li>
-
<li>Ligate 5 µl of the reaction mix with 50 ng EcoRI/PstI resticted and purified pSB1C3 over night at 16 °C <br> For Ligation use the T4-Ligase and fresh T4-Ligase Buffer with ATP</li>  
+
<li>Ligate 5 µl of the reaction mix with 50 ng EcoRI/PstI restricted and purified pSB1C3 over night at 16 °C <br> For Ligation use the T4-Ligase and fresh T4-Ligase Buffer with ATP</li>  
<li>After heat-inactivation of the ligase by 80 °C for 15 min use 2 µl of the mix for heat-shock transformation</li>
<li>After heat-inactivation of the ligase by 80 °C for 15 min use 2 µl of the mix for heat-shock transformation</li>
-
<li>Plate out the transformation in black petridishies  
+
<li>Plate out the transformation mix on black petridishies with LB-cmp media</li>
</ul>
</ul>
 +
<Br><Br><br>
-
<Br><Br><Br><Br><Br><Br><Br><Br>
+
<img alt="safety" src="/wiki/images/4/46/Bild6.png"  width="321" height="220" align="center">
 +
 
 +
<Br><Br><Br><Br><Br>
<font size="3" color="#F0F8FF" face="Arial regular">
<font size="3" color="#F0F8FF" face="Arial regular">
<p text-aligne:left style="margin-left:50px; margin-right:50px">
<p text-aligne:left style="margin-left:50px; margin-right:50px">
-
Construction of pSB1C3-petZ
 
-
For the construction of pSB1C3-petZ we isolate the petZ gene with PCR from the gBlocks C1 and C3:
+
<b><font size="5" color="#F0F8FF" face="Arial regular">Construction of pSB1C3-pezT</font></b>
 +
 
 +
<br>
 +
<br>
 +
For the construction of pSB1C3-pezT we isolated the pezT gene via PCR from the gBlocks C1, C2 and C3 following this protocol:
<br><br>
<br><br>
-
<img alt="Zeta_toxin_pcr" src="/wiki/images/c/c7/Zeta_toxin_pcr_-_psb1C3_-_psb1c3_restiction.jpg" width="200" height="200" align="right"style="margin-left:50px"; "margin-right:50px"></a>
+
 
 +
 
 +
 
 +
 
 +
 
 +
<dl class="igemTUD2013gelpicture2" style="margin-right:50px;">
 +
<dd><img alt="Zeta_toxin_pcr" src="/wiki/images/c/c7/Zeta_toxin_pcr_-_psb1C3_-_psb1c3_restiction.jpg" width="200" height="200" align="right"style="margin-left:50px"; "margin-right:50px"></dd>
 +
<dd><font size="2" color="#F0F8FF" face="Arial regular"><p align="justify">Fig. 2: M: 1kbp Ladder (Promega)<br>1: PCR isolated pezT gene. The pezT gene shows a signal at 700 bps <br>2: Unrestricted pSB1C3 <br>3: pSB1C3 cut by PstI and EcoRI. The empty plasmid shows a signal at 1.3 kbps</p></font></dd>
 +
</dl>
 +
 
 +
 
<ul style="margin-left:50px; margin-right:50px; text-align:justify; ">
<ul style="margin-left:50px; margin-right:50px; text-align:justify; ">
-
<li>Use the Primer pair Pre-PetZ/For-PetZ and an annealing temperature of 55 °C for the isolation PCR follow the Q5 PCR Protokol from NEB Biolabs.  </li>
+
<li>Use the primer pair Pre-PezT/For-PezT and an annealing temperature of 55 °C. For the isolation <br>PCR follow the Q5 PCR protocol from <a href="http://www.neb.com/"><font size="3" color="#F0F8FF" face="Arial regular"><b>NEB Biolabs</b></font></a>.  </li>
 +
<li> Use 5 µl for the control gel electrophoresis (1.2% agarose) and clean the rest of the reaction with <br>the Wizard SV Gel and PCR Clean-Up System from <a href="http://worldwide.promega.com/"><font size="3" color="#F0F8FF" face="Arial regular"><b>Promega</b></font></a>.</li>
 +
<li> Restrict 500 ng of the PCR product and 1 µg of pSB1C3 with EcoRI and PstI (10 U each) at 37 °C for 30 min. </li>
 +
<li> Perform the ligation with an molar ratio of 3:1 (insert:vector).<br> For Ligation use the T4-Ligase and fresh T4-Ligase Buffer with ATP</li>
 +
<li> Transform 2 µl of the ligation mix into <i>E. coli</i> DH5α. </li>
 +
<Li> Plate out the transformation on petri dishes with LB-cmp media.</li>
</ul>
</ul>
<br><br>
<br><br>
 +
 +
<img alt="safety" src="/wiki/images/0/0c/Psb1c3-petz_weiß2.png"  width="321" height="220" align="center">
 +
 +
<Br><Br><Br><Br><Br>
 +
 +
<b><font size="5" color="#F0F8FF" face="Arial regular"><p style="margin-left:50px"; align="left">Conclusion</p></font></b>
 +
 +
<Br>
 +
 +
<ul style="margin-left:50px; margin-right:50px; text-align:justify; ">
 +
 +
<li>The results from the gBlock assembly PCR (Fig. 1) could not be reproduced.</li>
 +
<li>The gBlock assembly PCR resulted in the right signal (Fig. 2). Nevertheless multiple transformations failed.</li>
 +
</ul>

Latest revision as of 01:56, 5 October 2013







Lab book | Materials | Protocols



Labjournal

gBlocks assembly

We designed the light induced kill switch based on the pDawn plasmid. We ordered 10 gBlock fragments coding for our plasmid from IDT. We assembeld the fragments according to the following protocol:

Assembly PCR with 10 gBlocks
Fig. 1:
M: GeneRuler DNA Ladder Mix (Thermo Sc.)

1: Assembly PCR. The signal at 5 kbps is our construct.

  • Reconstitute the gBlock fragments in 10 µl TE buffer
  • Use 1 µl of each gBlock fragment for a PCR with the Q5 Polymerase
  • Perform the PCR reaction with primers coding for the prefix and suffix and an annealing temperature of 55 °C
    (30 cycles)
  • Load an 0.8% agarose gel with 5 µl of PCR reaction (total volume 50 µl) and perform a DNA gel electrophoresis
  • Cut the other 45 µl with the restriction enzymes EcoRI and PstI (10 U each) for 1 h at 37 °C
  • Ligate 5 µl of the reaction mix with 50 ng EcoRI/PstI restricted and purified pSB1C3 over night at 16 °C
    For Ligation use the T4-Ligase and fresh T4-Ligase Buffer with ATP
  • After heat-inactivation of the ligase by 80 °C for 15 min use 2 µl of the mix for heat-shock transformation
  • Plate out the transformation mix on black petridishies with LB-cmp media



safety




Construction of pSB1C3-pezT

For the construction of pSB1C3-pezT we isolated the pezT gene via PCR from the gBlocks C1, C2 and C3 following this protocol:

Zeta_toxin_pcr

Fig. 2: M: 1kbp Ladder (Promega)
1: PCR isolated pezT gene. The pezT gene shows a signal at 700 bps
2: Unrestricted pSB1C3
3: pSB1C3 cut by PstI and EcoRI. The empty plasmid shows a signal at 1.3 kbps

  • Use the primer pair Pre-PezT/For-PezT and an annealing temperature of 55 °C. For the isolation
    PCR follow the Q5 PCR protocol from NEB Biolabs.
  • Use 5 µl for the control gel electrophoresis (1.2% agarose) and clean the rest of the reaction with
    the Wizard SV Gel and PCR Clean-Up System from Promega.
  • Restrict 500 ng of the PCR product and 1 µg of pSB1C3 with EcoRI and PstI (10 U each) at 37 °C for 30 min.
  • Perform the ligation with an molar ratio of 3:1 (insert:vector).
    For Ligation use the T4-Ligase and fresh T4-Ligase Buffer with ATP
  • Transform 2 µl of the ligation mix into E. coli DH5α.
  • Plate out the transformation on petri dishes with LB-cmp media.


safety




Conclusion
  • The results from the gBlock assembly PCR (Fig. 1) could not be reproduced.
  • The gBlock assembly PCR resulted in the right signal (Fig. 2). Nevertheless multiple transformations failed.








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