Team:TU Eindhoven/Notebook/Timeline

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                 <div class="col_700_2 float_r">
                 <div class="col_700_2 float_r">
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<h1>June</h1>
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<h3>Wetlab</h3>
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<body>
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<h2>Timeline of labwork</h2>
 +
<p>
 +
Click below to jump to a specific month:</p>
 +
<ul>
 +
  <li><a href="#June">June</a></li>
 +
  <li><a href="#July">July</a></li>
 +
  <li><a href="#August">August</a></li>
 +
  <li><a href="#September">September</a></li>
 +
  <li><a href="#October">October</a></li>
 +
</ul>
 +
<br />
 +
<h2 id="June">June</h2>
 +
<h4>Wetlab</h4>
<ul>
<ul>
   <li> Arrival of the biobricks</li>
   <li> Arrival of the biobricks</li>
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   <li>Amplification of PET 29a(+), pEVOL pAzF, BBa_K811005(Penn) &amp; CPX</li>
   <li>Amplification of PET 29a(+), pEVOL pAzF, BBa_K811005(Penn) &amp; CPX</li>
</ul>
</ul>
-
<h1>July</h1>
+
<h2 id="July">July</h2>
-
<h2>Week 27: June  30 – July 6</h2>
+
<h3>Week 27: June  30 – July 6</h3>
-
<h3>Wetlab</h3>
+
<h4>Wetlab</h4>
-
 
+
<ul>
<ul>
   <li>Plasmid amplification and  culturing BBa_K811005(Penn) + CPX  </li>
   <li>Plasmid amplification and  culturing BBa_K811005(Penn) + CPX  </li>
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</ul>
</ul>
-
   <h3>Microfluidics lab</h3>
+
   <h4>Microfluidics lab</h4>
<ul>
<ul>
   <li>Photo lithography</li>
   <li>Photo lithography</li>
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   <li> New polyacrylamide devices: Photo  lithography</li>
   <li> New polyacrylamide devices: Photo  lithography</li>
   <li> Improved protocol with shorter ashing  time, aquapel coating improvement, shorter baking time PDMS.</li></ul>
   <li> Improved protocol with shorter ashing  time, aquapel coating improvement, shorter baking time PDMS.</li></ul>
-
<h3>Week 28: July 7 – July 13</h3>
+
<h4>Week 28: July 7 – July 13</h4>
-
   <h3><br />
+
   <h4>
-
     Wetlab</h3>
+
     Wetlab</h4>
   <ul>
   <ul>
     <li>
     <li>
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     <li> Site directed mutagenesis of CPX </li>
     <li> Site directed mutagenesis of CPX </li>
   </ul>
   </ul>
-
   <h3>Microfluidics lab</h3>
+
   <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li>  Create oil and water phase, testing  devices in ICMS lab</li>
       <li>  Create oil and water phase, testing  devices in ICMS lab</li>
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       <li> Droplets separation (centrifuge) and  view with microscope</li>
       <li> Droplets separation (centrifuge) and  view with microscope</li>
     </ul>
     </ul>
-
     <h2>Week 29: July  14 – July 20</h2>
+
     <h3>Week 29: July  14 – July 20</h3>
-
     <h3>Wetlab</h3>
+
     <h4>Wetlab</h4>
     <ul>
     <ul>
       <li>  Site directed mutagenesis of CPX</li>
       <li>  Site directed mutagenesis of CPX</li>
       <li> Sequencing of CPX and BBa_K811005(Penn)</li>
       <li> Sequencing of CPX and BBa_K811005(Penn)</li>
     </ul>
     </ul>
-
     <h3>Microfluidics lab</h3>
+
     <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li>  Create oil and water phase</li>
       <li>  Create oil and water phase</li>
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       <li> Droplets separation (centrifuge) and  view with microscope</li>
       <li> Droplets separation (centrifuge) and  view with microscope</li>
     </ul>
     </ul>
-
     <h2>Week 30: July  21 – July 27</h2>
+
     <h3>Week 30: July  21 – July 27</h3>
-
     <h3>Wetlab</h3>
+
     <h4>Wetlab</h4>
     <ul>
     <ul>
       <li>    Sequencing results came  in and were as expected</li>
       <li>    Sequencing results came  in and were as expected</li>
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       <li> FACS –  DBCO PEG4 5/6 TAMRA </li>
       <li> FACS –  DBCO PEG4 5/6 TAMRA </li>
     </ul>
     </ul>
-
     <h3>Microfluidics lab</h3>
+
     <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li>  Soft lithography (glass binding)</li>
       <li>  Soft lithography (glass binding)</li>
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       <li> Photo lithography (no height  measurement + silenization) </li>
       <li> Photo lithography (no height  measurement + silenization) </li>
     </ul>
     </ul>
-
     <h2>Week 31: July  28 – August 3</h2>
+
     <h3>Week 31: July  28 – August 3</h3>
-
     <h3>Wetlab</h3>
+
     <h4>Wetlab</h4>
     <ul>
     <ul>
       <li>  Protein expression</li>
       <li>  Protein expression</li>
       <li> FACS –  DBCO PEG10kDa </li>
       <li> FACS –  DBCO PEG10kDa </li>
     </ul>
     </ul>
-
     <h3>Microfluidics lab</h3>
+
     <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li>  Photo lithography (height measurement  + silenization) </li>
       <li>  Photo lithography (height measurement  + silenization) </li>
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       <li>        Droplets separation (centrifuge) and  view with microscope</li>
       <li>        Droplets separation (centrifuge) and  view with microscope</li>
     </ul>
     </ul>
-
     <h1>August</h1>
+
     <h2 id="August">August</h2>
-
     <h2>Week 32: August  4 – August 10</h2>
+
     <h3>Week 32: August  4 – August 10</h3>
-
     <h3>Wetlab</h3>
+
     <h4>Wetlab</h4>
     <ul>
     <ul>
       <li>  Protein expression</li>
       <li>  Protein expression</li>
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       <li> Protein  expression curve </li>
       <li> Protein  expression curve </li>
     </ul>
     </ul>
-
     <h3>Microfluidics lab</h3>
+
     <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li>  Control polyacrylamide droplet with  Leica microscope and with phantom V9</li>
       <li>  Control polyacrylamide droplet with  Leica microscope and with phantom V9</li>
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       <li> Polyacrylamide droplets</li>
       <li> Polyacrylamide droplets</li>
     </ul>
     </ul>
-
     <h2>Week 33: August  11 – August 17</h2>
+
     <h3>Week 33: August  11 – August 17</h3>
-
     <h3>Wetlab</h3>
+
     <h4>Wetlab</h4>
     <ul>
     <ul>
       <li>  FACS  DBCO PEG 5kDa fluorescent&nbsp;</li>
       <li>  FACS  DBCO PEG 5kDa fluorescent&nbsp;</li>
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       <li> Primers ordered for biobricking</li>
       <li> Primers ordered for biobricking</li>
     </ul>
     </ul>
-
     <h3>Microfluidics lab</h3>
+
     <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li>  Control polyacrylamide droplet with  Leica microscope and with phantom V9</li>
       <li>  Control polyacrylamide droplet with  Leica microscope and with phantom V9</li>
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       <li> Photo lithography (aquapel and glass  binding)</li>
       <li> Photo lithography (aquapel and glass  binding)</li>
     </ul>
     </ul>
-
     <h2><br />
+
     <h3><br />
       Week 34: August 18 – August 24<br />
       Week 34: August 18 – August 24<br />
-
     </h2>
+
     </h3>
-
     <h3>Wetlab </h3>
+
     <h4>Wetlab </h4>
     <ul>
     <ul>
       <li>  Side directed mutagenesis COMPx  (BBa_K1492000) to mutate PSTI out of the sequence without changing the amino  acid sequence</li>
       <li>  Side directed mutagenesis COMPx  (BBa_K1492000) to mutate PSTI out of the sequence without changing the amino  acid sequence</li>
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       <li> We did not see a crispy band at the lane of the tRNA synthetase but we did  see a clear crispy band at the lane of COMPy.</li>
       <li> We did not see a crispy band at the lane of the tRNA synthetase but we did  see a clear crispy band at the lane of COMPy.</li>
     </ul>
     </ul>
-
     <h3>Microfluidics lab</h3>
+
     <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li>  Droplets separation (centrifuge) and  view with microscope in ML II lab in Helix)</li>
       <li>  Droplets separation (centrifuge) and  view with microscope in ML II lab in Helix)</li>
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       <li> Cutting and ashing the new devices</li>
       <li> Cutting and ashing the new devices</li>
     </ul>
     </ul>
-
     <h2>Week 35: August 25 – August 31 </h2>
+
     <h3>Week 35: August 25 – August 31 </h3>
-
     <h3>Wetlab</h3>
+
     <h4>Wetlab</h4>
     <ul>
     <ul>
       <li>  Ligase of PSB1C3 with COMPy</li>
       <li>  Ligase of PSB1C3 with COMPy</li>
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       <li> Sequencing results gave another  negative result</li>
       <li> Sequencing results gave another  negative result</li>
     </ul>
     </ul>
-
     <h3>Microfluidics lab</h3>
+
     <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li>  Treat devices with aquapel. After  baking some debris was found in the devices</li>
       <li>  Treat devices with aquapel. After  baking some debris was found in the devices</li>
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       <li> Continuing optimizing flow rates for  bead encapsulation. A different ratio of flow rates was used of W1:W2:O of  1:1:0.5</li>
       <li> Continuing optimizing flow rates for  bead encapsulation. A different ratio of flow rates was used of W1:W2:O of  1:1:0.5</li>
     </ul>
     </ul>
-
     <h1>September</h1>
+
     <h2 id="September">September</h2>
-
     <h2>Week 36: September  1 – September 7</h2>
+
     <h3>Week 36: September  1 – September 7</h3>
-
     <h3>Wetlab</h3>
+
     <h4>Wetlab</h4>
     <ul>
     <ul>
       <li>  Performed another Side directed  mutagenesis COMPx  to mutate PSTI out of  the sequence without changing the amino acid sequence</li>
       <li>  Performed another Side directed  mutagenesis COMPx  to mutate PSTI out of  the sequence without changing the amino acid sequence</li>
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       <li> Synthesis and purification of DNA  DBCO PEG4 product</li>
       <li> Synthesis and purification of DNA  DBCO PEG4 product</li>
     </ul>
     </ul>
-
     <h3>Microfluidics lab</h3>
+
     <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li>  PDMS treatment</li>
       <li>  PDMS treatment</li>
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       <li> Bead encapsulation. Stiff devices  causing cracks resulting in loose tubing</li>
       <li> Bead encapsulation. Stiff devices  causing cracks resulting in loose tubing</li>
     </ul>
     </ul>
-
     <h2>Week 37: September  8 – September 14</h2>
+
     <h3>Week 37: September  8 – September 14</h3>
-
     <h3>Wetlab</h3>
+
     <h4>Wetlab</h4>
     <ul>
     <ul>
       <li>  Sequencing result showed a successful  mutation, however due to an error in the primer another mutation was  introduced. However we chose to continue with this gene for overhang PCR and do  another side directed mutagenesis at the end</li>
       <li>  Sequencing result showed a successful  mutation, however due to an error in the primer another mutation was  introduced. However we chose to continue with this gene for overhang PCR and do  another side directed mutagenesis at the end</li>
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       <li> Cell viability test 1</li>
       <li> Cell viability test 1</li>
     </ul>
     </ul>
-
     <h3>Microfluidics lab</h3>
+
     <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li>  PDMS pouring on the wafer. Bake PDMS  for 1.15 h for more flexible devices</li>
       <li>  PDMS pouring on the wafer. Bake PDMS  for 1.15 h for more flexible devices</li>
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       <li> Tests with bead encapsulation  successful. Fluorescent pictures obtained however fluorescent signal intensity  of single beads was too low to be detected</li>
       <li> Tests with bead encapsulation  successful. Fluorescent pictures obtained however fluorescent signal intensity  of single beads was too low to be detected</li>
     </ul>
     </ul>
-
     <h2>Week 38: September  15 – September 21</h2>
+
     <h3>Week 38: September  15 – September 21</h3>
-
     <h3>Wetlab</h3>
+
     <h4>Wetlab</h4>
     <ul>
     <ul>
       <li>  We chose to culture the vector of a  previously ordered biobrick we used for our project. (BBA_K811005) To make sure  we had enough vector for our ligation</li>
       <li>  We chose to culture the vector of a  previously ordered biobrick we used for our project. (BBA_K811005) To make sure  we had enough vector for our ligation</li>
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       <li> Testing of Rolling Circle and Multiple  Chain Amplification without cells on agarose gel</li>
       <li> Testing of Rolling Circle and Multiple  Chain Amplification without cells on agarose gel</li>
     </ul>
     </ul>
-
     <h3>Microfluidics lab</h3>
+
     <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li>  Soft lithography of PDMS. Made two  batches (= 10 devices)</li>
       <li>  Soft lithography of PDMS. Made two  batches (= 10 devices)</li>
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       <li> Try washing used devices to remove  beads. No movement observed. Beads still coagulated in other devices</li>
       <li> Try washing used devices to remove  beads. No movement observed. Beads still coagulated in other devices</li>
     </ul>
     </ul>
-
     <h2>Week 39: September  22 – September 28</h2>
+
     <h3>Week 39: September  22 – September 28</h3>
-
     <h3>Wetlab</h3>
+
     <h4>Wetlab</h4>
     <ul>
     <ul>
       <li>  Ligation of tRNA synthetase, COMPy and  COMPx into PSB1C3</li>
       <li>  Ligation of tRNA synthetase, COMPy and  COMPx into PSB1C3</li>
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       <li> Testing of Rolling Circle and Multiple  Chain Amplification without cells on agarose gel</li>
       <li> Testing of Rolling Circle and Multiple  Chain Amplification without cells on agarose gel</li>
     </ul>
     </ul>
-
     <h3>Microfluidics lab</h3>
+
     <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li>  Photo lithography</li>
       <li>  Photo lithography</li>
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       <li> Testing devices with fluorescent  bacteria.</li>
       <li> Testing devices with fluorescent  bacteria.</li>
     </ul>
     </ul>
-
     <h1>October</h1>
+
     <h2 id="October">October</h2>
-
     <h2>Week 40: September  29 – September 5</h2>
+
     <h3>Week 40: September  29 – September 5</h3>
-
     <h3>Wetlab</h3>
+
     <h4>Wetlab</h4>
     <ul>
     <ul>
       <li>  Side directed mutagenesis to correct  the mutated amino acid in the PSTI mutation</li>
       <li>  Side directed mutagenesis to correct  the mutated amino acid in the PSTI mutation</li>
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       <li> Cell viability test 2</li>
       <li> Cell viability test 2</li>
     </ul>
     </ul>
-
     <h3>Microfluidics lab</h3>
+
     <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li>  Small culture</li>
       <li>  Small culture</li>
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       <li> Testing devices with fluorescent  bacteria. Bacteria were visible and devices were working. It was hard to  stabilize the flow</li>
       <li> Testing devices with fluorescent  bacteria. Bacteria were visible and devices were working. It was hard to  stabilize the flow</li>
     </ul>
     </ul>
-
     <h2>Week 41: October  6 – October 12</h2>
+
     <h3>Week 41: October  6 – October 12</h3>
-
     <h3>Wetlab</h3>
+
     <h4>Wetlab</h4>
     <ul>
     <ul>
       <li>  Sequencing results were successful and  positive</li>
       <li>  Sequencing results were successful and  positive</li>
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       <li> Cell viability test 3</li>
       <li> Cell viability test 3</li>
     </ul>
     </ul>
-
     <h3>Microfluidics lab</h3>
+
     <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li>  Cut and ash devices. Ashing protocol  adjusted to RF power: 30 W; vent time: 60 s; bleed time: 20 s.W at a pressure  of 0.9 mbar</li>
       <li>  Cut and ash devices. Ashing protocol  adjusted to RF power: 30 W; vent time: 60 s; bleed time: 20 s.W at a pressure  of 0.9 mbar</li>
Line 395: Line 522:
       <li> Droplet encapsulation was tested with  polyacrylamide devices. Droplets were formed successfully. A new oil phase was  made due to breaking of the droplets to ensure the quality of the oil phase.  After this the problem was solved.</li>
       <li> Droplet encapsulation was tested with  polyacrylamide devices. Droplets were formed successfully. A new oil phase was  made due to breaking of the droplets to ensure the quality of the oil phase.  After this the problem was solved.</li>
     </ul>
     </ul>
-
     <h2>Week 42: October  13 – October 19</h2>
+
     <h3>Week 42: October  13 – October 19</h3>
-
     <h3>Microfluidics lab</h3>
+
     <h4>Microfluidics lab</h4>
     <ul>
     <ul>
       <li> Start small culture.</li>
       <li> Start small culture.</li>
       <li>Start protein expression.</li>
       <li>Start protein expression.</li>
-
       <li>Droplet encapsulation was tested with  polyacrylamide devices and fluorescence bacteria</li>
+
       <li>Droplet encapsulation was tested with  polyacrylamide devices and fluorescent bacteria</li>
     </ul>
     </ul>
   </li>
   </li>

Latest revision as of 00:57, 18 October 2014

iGEM Team TU Eindhoven 2014

iGEM Team TU Eindhoven 2014

Untitled Document

Timeline of labwork

Click below to jump to a specific month:


June

Wetlab

  • Arrival of the biobricks
  • The designs for the plasmids are approved and ordered Primers
  • Preparation of culture media, agar plates, antibiotics and glycerol 
  • Amplification of PET 29a(+), pEVOL pAzF, BBa_K811005(Penn) & CPX

July

Week 27: June 30 – July 6

Wetlab

  • Plasmid amplification and culturing BBa_K811005(Penn) + CPX
  • Vector and insert Digestions
  • BBa_K811005(Penn) + CPX insert ligate pET29a 
  • Colony PCR

Microfluidics lab

  • Photo lithography
  • Soft lithography
  • Testing polyacrylamide device with dye
  • Protocol aquapel
  • New polyacrylamide devices: Photo lithography
  • Improved protocol with shorter ashing time, aquapel coating improvement, shorter baking time PDMS.

Week 28: July 7 – July 13

Wetlab

  • Colony PCR
  • Site directed mutagenesis of CPX

Microfluidics lab

  • Create oil and water phase, testing devices in ICMS lab
  • Photo lithography (height measurement + silanization)
  • Control polyacrylamide droplet with Leica microscope and with phantom V9
  • Droplets separation (centrifuge) and view with microscope

Week 29: July 14 – July 20

Wetlab

  • Site directed mutagenesis of CPX
  • Sequencing of CPX and BBa_K811005(Penn)

Microfluidics lab

  • Create oil and water phase
  • Control polyacrylamide droplet with Leica microscope and with phantom V9
  • Droplets separation (filtration) and view with microscope
  • Soft lithography (no glass binding)
  • Soft lithography (glass binding)
  • Protocol aquapel
  • Create oil and water phase
  • Droplets separation (centrifuge) and view with microscope

Week 30: July 21 – July 27

Wetlab

  • Sequencing results came in and were as expected
  • Protein expression
  • FACS – DBCO PEG4 5/6 TAMRA

Microfluidics lab

  • Soft lithography (glass binding)
  • Protocol aquapel
  • Control polyacrylamide droplet with Leica microscope and with phantom V9
  • Photo lithography (no height measurement + silenization)

Week 31: July  28 – August 3

Wetlab

  • Protein expression
  • FACS – DBCO PEG10kDa

Microfluidics lab

  • Photo lithography (height measurement + silenization)
  • Soft lithography (glass binding)
  • Treat polyacrylamide and cell encapsulation devices with aquapel
  • Control polyacrylamide droplet with Leica microscope and with phantom V9
  • Droplets separation (centrifuge) and view with microscope

August

Week 32: August 4 – August 10

Wetlab

  • Protein expression
  • FACS – Antibody titration
  • Protein expression curve

Microfluidics lab

  • Control polyacrylamide droplet with Leica microscope and with phantom V9
  • Protocol aquapel
  • Droplets separation (centrifuge) and view with microscope
  • Photo lithography
  • Polyacrylamide droplets

Week 33: August 11 – August 17

Wetlab

  • FACS DBCO PEG 5kDa fluorescent 
  • Primers designed for biobricking
  • Primers ordered for biobricking

Microfluidics lab

  • Control polyacrylamide droplet with Leica microscope and with phantom V9
  • Photo lithography (no aquapel and no glass binding)
  • Droplets separation (centrifuge) and view with microscope
  • Photo lithography (aquapel and glass binding)


Week 34: August 18 – August 24

Wetlab

  • Side directed mutagenesis COMPx (BBa_K1492000) to mutate PSTI out of the sequence without changing the amino acid sequence
  • Overhang PCR tRNA synthetase (BBa_K1492002) & COMPy (BBa_K1492001)
  • Digestion PCR linearized backbone PSB1C3
  • COMPx sent for sequencing
  • Running agarose gel with tRNA synthetase and COMPy
  • We did not see a crispy band at the lane of the tRNA synthetase but we did see a clear crispy band at the lane of COMPy.

Microfluidics lab

  • Droplets separation (centrifuge) and view with microscope in ML II lab in Helix)
  • Control polyacrylamide droplet with Leica microscope and with phantom V9
  • Droplet separation by adding first HFE 7500 and then PFO to gain better control over the two phases
  • Wafer production of cell encapsulation devices. After wafer was developed PDMS treatment was performed with 60 g PDMS instead of 50 g
  • Cutting and ashing the new devices

Week 35: August 25 – August 31

Wetlab

  • Ligase of PSB1C3 with COMPy
  • Performed again overhang PCR for tRNA synthetase
  • Sequencing results COMPx gave a negative result so we send another 5 samples for sequencing
  • Running agarose gel with tRNA synthetase
  • We did not see a crispy band at the lane of the tRNA synthetase again
  • Performing colony PCR on the ligation product. The results showed an unsuccessful ligation
  • Sequencing results gave another negative result

Microfluidics lab

  • Treat devices with aquapel. After baking some debris was found in the devices
  • Droplet formation with new devices to optimize the flow rates for bead encapsulation later. The flow rates were adjusted accordingly to W1:W2:O of 2:2:0.5
  • Continuing optimizing flow rates for bead encapsulation. A different ratio of flow rates was used of W1:W2:O of 1:1:0.5

September

Week 36: September 1 – September 7

Wetlab

  • Performed another Side directed mutagenesis COMPx  to mutate PSTI out of the sequence without changing the amino acid sequence
  • Performed another colony PCR on the ligation product. The results showed an unsuccessful ligation again
  • Checked the primers for the overhang of the tRNA synthetase and COMPy again. Due to a miscommunication wrong primers were designed we found out
  • Designed new primers for COMPx, COMPy and the tRNA synthetase
  • Ordered new linear backbone PSB1C3
  • Synthesis and purification of DNA DBCO PEG4 product

Microfluidics lab

  • PDMS treatment
  • Fluorescent beads finally arrived. Cutting and ashing devices
  • Aquapel treatment of the devices
  • Encapsulation of beads
  • Some devices seemed to be too stiff causing cracks in the PDMS which resulted in poor attachment of tubing
  • Bead encapsulation. Stiff devices causing cracks resulting in loose tubing

Week 37: September 8 – September 14

Wetlab

  • Sequencing result showed a successful mutation, however due to an error in the primer another mutation was introduced. However we chose to continue with this gene for overhang PCR and do another side directed mutagenesis at the end
  • Primers and backbone we ordered arrived
  • Overhang PCR of tRNA synthetase, COMPy and COMPx
  • Digestion of linearized backbone
  • Digestion of PCR products
  • Reacting DNA DBCO with cells and testing reaction using FACS
  • Creating circular DNA template for use in Rolling Circle Amplification
    Protein expression
  • Cell viability test 1

Microfluidics lab

  • PDMS pouring on the wafer. Bake PDMS for 1.15 h for more flexible devices
  • Cut PDMS and ash devices
  • Treat devices with Aquapel
  • Could not run tests due to maintenance of the microscope
  • Tests with bead encapsulation successful. Fluorescent pictures obtained however fluorescent signal intensity of single beads was too low to be detected

Week 38: September 15 – September 21

Wetlab

  • We chose to culture the vector of a previously ordered biobrick we used for our project. (BBA_K811005) To make sure we had enough vector for our ligation
  • Running agarose gel of tRNA synthetase, COMPy and COMPx. This time we saw nice crispy bands at the right place
  • Mini prepping, digestion and gel extraction of the cultured vector PSB1C3
  • Second attempt at creating circular template (previous product was impure)
  • Testing of Rolling Circle and Multiple Chain Amplification without cells on agarose gel

Microfluidics lab

  • Soft lithography of PDMS. Made two batches (= 10 devices)
  • Cut PDMS and ash devices
  • Aquapel devices. Two devices showed to have debris left after aquapeling
  • Bead encapsulation. Beads coagulated in the channels
  • Try washing used devices to remove beads. No movement observed. Beads still coagulated in other devices

Week 39: September 22 – September 28

Wetlab

  • Ligation of tRNA synthetase, COMPy and COMPx into PSB1C3
  • Colony picking of ligation product. For every insert we had a positive result
  • Vectors sent for sequencing
  • Testing of Rolling Circle and Multiple Chain Amplification without cells on agarose gel

Microfluidics lab

  • Photo lithography
  • No height measurement due to broken equipment in lab
  • Soft lithography, PDMS treatment and baking
  • Cut devices and ash. Three out of five devices were usable due to bad oxygen ashing
  • Start small culture
  • Treat devices with aquapel. No dirt seemed to be visible after drying with pressurized nitrogen
  • Start protein expression. First OD measurement was 0.953. Continued inducing with IPTG
  • After protein expression OD was 0.343 which equals 2.74*108 cells/mL
  • Testing devices with fluorescent bacteria.

October

Week 40: September 29 – September 5

Wetlab

  • Side directed mutagenesis to correct the mutated amino acid in the PSTI mutation
  • Sequencing results were successful and positive
  • Mutated vector send for sequencing
  • Testing of Rolling Circle Amplification without cells on agarose gel
  • Protein expression
  • Cell viability test 2

Microfluidics lab

  • Small culture
  • Protein expression
  • Testing devices with fluorescent bacteria. Bacteria were visible and devices were working. It was hard to stabilize the flow

Week 41: October 6 – October 12

Wetlab

  • Sequencing results were successful and positive
  • Biobrick sent on 8 october!
  • Testing Rolling Circle Amplification on cells, verify using FACS
  • Protein expression
  • Cell viability test 3

Microfluidics lab

  • Cut and ash devices. Ashing protocol adjusted to RF power: 30 W; vent time: 60 s; bleed time: 20 s.W at a pressure of 0.9 mbar
  • Start small culture
  • Treated devices with aquapel. One device appeared to show no binding with the glass. Start protein expression
  • OD after protein expression was 0.94. This equals to 1.5*1010 cells/mL in the stock solution. Due to maintenance of the microscope bacteria were stored till the next day
  • Droplet encapsulation was tested with polyacrylamide devices. Droplets were formed successfully. A new oil phase was made due to breaking of the droplets to ensure the quality of the oil phase. After this the problem was solved.

Week 42: October 13 – October 19

Microfluidics lab

  • Start small culture.
  • Start protein expression.
  • Droplet encapsulation was tested with polyacrylamide devices and fluorescent bacteria
iGEM Team TU Eindhoven 2014

Retrieved from "http://2014.igem.org/Team:TU_Eindhoven/Notebook/Timeline"