Team:UNIPV-Pavia/Calendar/August/week1

From 2011.igem.org

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<p><a name="indice"/>
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</p>
</p>
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<div align="justify">
<a name="August.2C_1st"></a><h2> <span class="mw-headline">August, 1st</span></h2>
<a name="August.2C_1st"></a><h2> <span class="mw-headline">August, 1st</span></h2>
<p>
<p>
-
For each plate from the previous day, two colonies were picked.
+
Two colonies for E28-AGAIN and E41-AGAIN plates were picked and inoculated in LB + Cm12.5; J101-31, J101-E5 and J101-E7 plasmids were extracted:
</p>
</p>
 +
 +
<center>
 +
<table class="data">
 +
    <tr>
 +
      <td class="row"><b>Plasmid</b></td>
 +
      <td class="row"><b>DNA (ng/&mu;l)</b></td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row">J101-31</td>
 +
      <td class="row">38</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row">J101-E5</td>
 +
      <td class="row">14.5</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row">J101-E7</td>
 +
      <td class="row">14</td>
 +
  </tr>
 +
 +
</table>
 +
</center>
<p>
<p>
-
Next, MGZ1 competent cells were prepared according to protocol. They were put in 22 100 &mu;l test-tubes.  
+
These plasmids were digested with EcoRI and SpeI restriction enzymes and screened by gel-electrophoresis; a medium-size agarose gel was then prepared.
 +
<br>
 +
MGZ1 competent cells were prepared according to protocol. They were put in 22 100 &mu;l test-tubes.
 +
<br>
 +
Gel electrophoresis was performed:
</p>
</p>
-
<p><a name="indice"/>
+
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV01_08_2011_J101-31_ES_J101-E5_ES-J101-E7_ES.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f8/UNIPV01_08_2011_J101-31_ES_J101-E5_ES-J101-E7_ES.png" class="thumbimage" height="80%" width="80%"></a> <div class="thumbcaption">Medium size gel</div></div></div></div>
-
</p>
+
-
<a name="August.2C_2nd"></a><h2> <span class="mw-headline">August, 2nd</span></h2>
+
<p>
<p>
 +
Insert lengths were all correct so J101-31, J101-E5, J101-E7 were transformed from MiniPrepped DNA in MGZ1 competent cells.
 +
</p> 
 +
<br>
 +
E37-2, E38-1, E39-1, E40-2 and E42-1 were inoculated in 5 ml LB + Cm12.5 and grown ON for plasmid extraction.
 +
</p>
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
<a name="August.2C_2nd"></a><h2> <span class="mw-headline">August, 2nd</span></h2>
<p>
<p>
-
Plasmids containing E28AGAIN-1, E28AGAIN-2, E41AGAIN-1, E28AGAIN-2, E37-2, E38-1. E39-1, E40-2 and E42-1 were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:
+
MGZ1 J101-31, J101-E5 and J101-E7 plates were all grown; one colony was picked from each of them and inoculated in LB + Cm12.5.
 +
Plasmids harboring E28-AGAIN-1, E28-AGAIN-2, E41-AGAIN-1, E28-AGAIN-2, E37-2, E38-1, E39-1, E40-2 and E42-1 were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:
</p>
</p>
<center>
<center>
-
<table border="1">
+
<table class="data">
     <tr>
     <tr>
-
       <td><b>Plasmid</b></td>
+
       <td class="row"><b>Plasmid</b></td>
-
       <td><b>DNA (ng/&mu;l)</b></td>
+
       <td class="row"><b>DNA (ng/&mu;l)</b></td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E28AGAIN-1</td>
+
       <td class="row">E28AGAIN-1</td>
-
       <td>21.4</td>
+
       <td class="row">21.4</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E28AGAIN-2</td>
+
       <td class="row">E28AGAIN-2</td>
-
       <td>21.0</td>
+
       <td class="row">21.0</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E41AGAIN-1</td>
+
       <td class="row">E41AGAIN-1</td>
-
       <td>33.7</td>
+
       <td class="row">33.7</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E41AGAIN-2</td>
+
       <td class="row">E41AGAIN-2</td>
-
       <td>27.7</td>
+
       <td class="row">27.7</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E37-2</td>
+
       <td class="row">E37-2</td>
-
       <td>18.5</td>
+
       <td class="row">18.5</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E38-1</td>
+
       <td class="row">E38-1</td>
-
       <td>26.0</td>
+
       <td class="row">26.0</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E39-1</td>
+
       <td class="row">E39-1</td>
-
       <td>21.6</td>
+
       <td class="row">21.6</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E40-2</td>
+
       <td class="row">E40-2</td>
-
       <td>19.9</td>
+
       <td class="row">19.9</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E42-1</td>
+
       <td class="row">E42-1</td>
-
       <td>25.1</td>
+
       <td class="row">25.1</td>
   </tr>
   </tr>
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<p>
<p>
-
J101-31, J101-E5, J101-E7 were transformed in MGZ1 competent cells.
+
Digestions of E28AGAIN-1, E28AGAIN-2, E41AGAIN-1 and E42AGAIN-2 were performed for screening; a 5x mix was prepared and for each sample the volumes specified in the table below were used:
-
</p>
+
</p>
 +
 
 +
<center>
 +
<table class="data">
 +
    <tr>
 +
      <td class="row"><b>DNA (&mu;l)</b></td>
 +
      <td class="row"><b>H<small><sub>2</sub></small>O (&mu;l)</b></td>
 +
      <td class="row"><b>Enzyme 1 (&mu;l)</b></td>
 +
      <td class="row"><b>Enzyme 2 (&mu;l)</b></td>
 +
      <td class="row"><b>Buffer H (&mu;l)</b></td>
 +
      <td class="row"><b>Final Volume (&mu;l)</b></td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td class="row">5</td>
 +
      <td class="row">16.5</td>
 +
      <td class="row">0.5 EcoRI</td>
 +
      <td class="row">0.5 PstI</td>
 +
      <td class="row">2.5</td>
 +
      <td class="row">25</td>
 +
  </tr>
 +
 
 +
</table>
 +
</center>
<p>
<p>
-
Digestions of E28AGAIN-1, E28AGAIN-2, E41AGAIN-1 and E42AGAIN-2 were performed for screening; a 5x mix was prepared and for each sample the volumes specified in the table below were used:
+
A small-size agarose gel was prepared for gel electrophoresis.
 +
<br>
 +
TOP10 competent cells, harboring <a href="http://partsregistry.org/wiki/index.php/Part:BBa_T9002">BBa_T9002</a> construct, were co-transformed with ENTERO4C5 to confer resistance to Cloramphenicol (12.5 mg/ml) and plated on LB agar + Amp + Cm 12.5 plates.
 +
<br>
 +
In the afternoon gel electrophoresis was performed:
</p>
</p>
 +
 +
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_02_08_2011_E41-AGAIN-1_EP_E41-AGAIN-2_EP_E28-AGAIN-1_EP_E28-AGAIN-2_EP.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/6f/UNIPV_02_08_2011_E41-AGAIN-1_EP_E41-AGAIN-2_EP_E28-AGAIN-1_EP_E28-AGAIN-2_EP.png" class="thumbimage" height="80%" width="80%"></a>  <div class="thumbcaption">Small size gel</div></div></div></div>
 +
 +
<p>
 +
Unfortunately insert length was correct only for E28-AGAIN-1 so we ligated again E41:
 +
</p>
 +
 +
<center>
 +
<table class="data">
 +
    <tr>
 +
      <td class="row"><b>Ligation Name</b></td>
 +
      <td class="row"><b>Vector</b></td>
 +
      <td class="row"><b>Vector volume (&mu;l)</b></td>
 +
      <td class="row"><b>Insert</b></td>
 +
      <td class="row"><b>Insert volume (&mu;l)</b></td>
 +
      <td class="row"><b>Buffer (&mu;l)</b></td>
 +
      <td class="row"><b>T4 Ligase (&mu;l)</b></td>
 +
  </tr>
 +
 +
    <tr>
 +
      <td class="row"><b>E41N</b></td>
 +
      <td class="row">E36 (S-P)</td>
 +
      <td class="row">4</td>
 +
      <td class="row">E3-1 (X-P)</td>
 +
      <td class="row">4</td>
 +
      <td class="row">1</td>
 +
      <td class="row">1</td>
 +
  </tr>
 +
</table>
 +
</center>
 +
 +
<p>
 +
Glycerol stocks were prepared according to the protocol for J101-31, J101-E5 and J101-E7.
 +
<br>
 +
E37-2, E38-1, E39-1, E40-2, E42-1 and E28-AGAIN-1 DNA was sent to BMR genomics for sequencing.
 +
<br>
 +
5 &mu;l of E37-2, E38.1, E39-1, E40-2 and ENTERO4C5 were inoculated in 1 ml M9 + Cm12.5 for a preliminary test on the autoinducer inactivation enzyme A (AiiA) enzyme.
 +
</p>
 +
 +
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
<a name="August.2C_3rd"></a><h2> <span class="mw-headline">August, 3rd</span></h2>
 +
 +
<p>
 +
T9002-ENTERO4C5 plate was grown and a colony was picked and inoculated in LB + Amp + Cm12.5; E41N ligation was transformed in 100 &mu;l of MGZ1 competent cells and plated on a LB + Cm12.5 plate.
 +
<br>
 +
34 LB agar + Cm12.5 plates were prepared according to protocols.
 +
<br>
 +
E37-2, E38.1, E39-1, E40-2 and ENTERO4C5 cultures were diluted 1:100 in a final volume of 5 ml of M9 + Cm12.5 for the preliminary test; a 1000 x 75 ng/&mu;l atc stock was prepared.
 +
<br>
 +
After 3 hours cultures were induced with 5 &mu;l of atc (75 ng/ml final concentration); after 4 hours 2.5 &mu;l of 2mM 3OC<sub><small>6</small></sub>-HSL (1:2000 dilution) were added (final 3OC<sub><small>6</small></sub>-HSL concentration 1&mu;M) and the experiment started (t = 0 h).
 +
<br>
 +
250 &mu;l samples were taken at t = 0 h, t = 1 h, and t = 4 h; O.D. at 600 nm was measured:
 +
</p>
 +
<center>
<center>
-
<table border="1">
+
<table class="data">
     <tr>
     <tr>
-
       <td><b>DNA (&mu;l)</b></td>
+
       <td class="row"></td>
-
       <td><b>H<small><sub>2</sub></small>O (&mu;l)</b></td>
+
       <td class="row"><b>E37</b></td>
-
       <td><b>Enzyme 1 (&mu;l)</b></td>
+
       <td class="row"><b>E38</b></td>
-
       <td><b>Enzyme 2 (&mu;l)</b></td>
+
       <td class="row"><b>E39</b></td>
-
       <td><b>Buffer H (&mu;l)</b></td>
+
       <td class="row"><b>E40</b></td>
-
       <td><b>Final Volume (&mu;l)</b></td>
+
       <td class="row"><b>ENTERO4C5</b></td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>5</td>
+
       <td class="row"><b>t = 0 h</b></td>
-
       <td>16.5</td>
+
       <td class="row">0.12</td>
-
       <td>0.5 EcoRI</td>
+
       <td class="row">0.11</td>
-
       <td>0.5 Pstl</td>
+
       <td class="row">0.13</td>
-
       <td>2.5</td>
+
       <td class="row">0.10</td>
-
       <td>25</td>
+
       <td class="row">0.11</td>
   </tr>
   </tr>
 +
 +
  <tr>
 +
      <td class="row"><b>t = 1 h</b></td>
 +
      <td class="row">0.22</td>
 +
      <td class="row">0.15</td>
 +
      <td class="row">0.21</td>
 +
      <td class="row">0.28</td>
 +
      <td class="row">0.19</td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row"><b>t = 4 h</b></td>
 +
      <td class="row">0.47</td>
 +
      <td class="row">0.60</td>
 +
      <td class="row">0.53</td>
 +
      <td class="row">0.37</td>
 +
      <td class="row">0.53</td>
 +
  </tr>
 +
</table>
</table>
</center>
</center>
 +
<p>
 +
Then samples were centrifuged at 13.000 rpm; finally the supernatant was collected and stored at -20°C.
 +
<br>
 +
Glycerol stock for T9002-ENTERO4C5 was prepared.
 +
<br>
 +
Two 5 &mu;l inocula of E32, E33, E34, E35, J101-E5, J101-31, J101-E6, J101-E7 and ENTERO4C5 were prepared in 1 ml of M9 + Cm12.5 for a preliminary test on TetR repressible promoter.
 +
</p>
 +
 +
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
<a name="August.2C_4th"></a><h2> <span class="mw-headline">August, 4th</span></h2>
<p>
<p>
-
TOP10 competent cells, harboring <a href="http://partsregistry.org/wiki/index.php/Part:BBa_T9002">BBa_T9002</a> construct, were co-transformed with ENTERO4C5 to confer resistance to Cloramphenicol (12.5 mg/ml).
+
E41N plate was grown; three colonies were picked (E41N-1, E41N-2, E41N-3) and inoculated in LB + Cm12.5.
 +
<br>
 +
E32-1, E33-1, E34-1, E35-1, J101-E5-1, J101-31-1, J101-E6-1, J101-E7-1, E32-2, E33-2, E34-2, E35-2, J101-E5-2, J101-31-2, J101-E6-2, J101-E7-2, ENTERO4C5-1 and ENTERO4C5-2 were diluted 1:500 in a final volume of 1 ml.
 +
After 2 hours and 30 minutes inducible cultures were supplemented with anhydrotetracyclin (atc) at final concentrations of: 1 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml. Moreover uninduced cultures were supplemented with ddH<small><sub>2</sub></small>O.
 +
<br>
 +
ENTERO-RBS and T9002-ENTERO4C5 were inoculated in 1 ml of Cm12.5 for preliminary AiiA test (with supernatants collected on August, 3rd).
</p>
</p>
-
<table border="0" width="100%" class="menu">
+
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
-
<tr>
+
<a name="August.2C_5th"></a><h2> <span class="mw-headline">August, 5th</span></h2>
-
<td align="left"><a href="/Team:UNIPV-Pavia/Calendar/July/settimana5" title="Team:UNIPV-Pavia/Calendar/JuLY/settimana5"> Previous week</a></td>
+
<p>
-
<td align="right"><a href="/Team:UNIPV-Pavia/Calendar/August/week2" title="Team:UNIPV-Pavia/Calendar/August/week2"> Next week</a></td>
+
ENTERO-RBS and T9002-ENTERO4C5 cultures were diluted 1:500 in a final volume of 1 ml and 10 ml of M9 + Cm12.5 respectively; they were grown for six hours. Plasmid purification was performed for E41N-1, E41N-2 and E41N-3:
-
</tr>
+
</p>
 +
 
 +
<center>
 +
<table class="data">
 +
    <tr>
 +
      <td class="row"><b>Plasmid</b></td>
 +
      <td class="row"><b>DNA (ng/&mu;l)</b></td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td class="row">E41N-1</td>
 +
      <td class="row">62.3</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td class="row">E41N-2</td>
 +
      <td class="row">17.3</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td class="row">E41N-3</td>
 +
      <td class="row">43.9</td>
 +
  </tr>
</table>
</table>
 +
</center>
 +
<p>
 +
Screening digestion with EcoRI and PstI restriction enzymes was carried out:
 +
</p>
 +
 +
<table class="data">
 +
    <tr>
 +
      <td class="row"><b>Plasmid</b></td>
 +
      <td class="row"><b>DNA (&mu;l)</b></td>
 +
      <td class="row"><b>H<small><sub>2</sub></small>O (&mu;l)</b></td>
 +
      <td class="row"><b>Enzyme 1 (&mu;l)</b></td>
 +
      <td class="row"><b>Enzyme 2 (&mu;l)</b></td>
 +
      <td class="row"><b>Buffer H (&mu;l)</b></td>
 +
      <td class="row"><b>Final Volume (&mu;l)</b></td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row"></html>E41N-1<html></td>
 +
      <td class="row"></html>2.5<html></td>
 +
      <td class="row"></html>19<html></td>
 +
      <td class="row">0.5 EcoRI</td>
 +
      <td class="row">0.5 Pstl</td>
 +
      <td class="row">2.5</td>
 +
      <td class="row">25</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row"></html>E41N-2<html></td>
 +
      <td class="row"></html>8.5<html></td>
 +
      <td class="row"></html>13<html></td>
 +
      <td class="row">0.5 EcoRI</td>
 +
      <td class="row">0.5 Pstl</td>
 +
      <td class="row">2.5</td>
 +
      <td class="row">25</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row"></html>E41N-2<html></td>
 +
      <td class="row"></html>3.5<html></td>
 +
      <td class="row"></html>18<html></td>
 +
      <td class="row">0.5 EcoRI</td>
 +
      <td class="row">0.5 Pstl</td>
 +
      <td class="row">2.5</td>
 +
      <td class="row">25</td>
 +
  </tr>
 +
 +
</table>
 +
</center>
 +
 +
<p>
 +
3OC<sub><small>6</small></sub>-HSL was diluted to known concentrations in order to induce T9002-ENTERO4C5 biosensor and obtain a reference curve for the measurements on E37, E38, E39, E40 and ENTERO4C5 supernatants.
 +
<br>
 +
A small-size agarose gel was prepared; in the afternoon gel electrophoresis was carried out:
 +
</p>
 +
 +
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_05_08_2011_E41N1_EP_E41N2_EP_E41N3_EP.pn" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c9/UNIPV_05_08_2011_E41N1_EP_E41N2_EP_E41N3_EP.png" class="thumbimage" height="80%" width="80%"></a>  <div class="thumbcaption">Small size gel</div></div></div></div>
 +
 +
<p>
 +
Only E41N-1 showed the correct band, so we decided to keep it. E41N-2 and E41N-3 glycerol stocks were thrown.
 +
<br>
 +
TECAN test on E37, E38, E39, E40 and ENTERO4C5 supernatants was carried out; as supernatants were not diluted enough it was not possible to precisely measure 3OC<sub><small>6</small></sub>-HSL concentration as the biosensor (T9002-ENTERO4C5) worked in the saturation zone.
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Latest revision as of 10:02, 18 September 2011

UNIPV TEAM 2011

March
M T W T F S S
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7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31    

April
M T W T F S S
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4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30

May
M T W T F S S
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2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 31

June
M T W T F S S
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6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30

July
M T W T F S S
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4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31

August
M T W T F S S
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31

September
M T W T F S S
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5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30

October
M T W T F S S
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3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31

AUGUST: WEEK 1

August, 1st

Two colonies for E28-AGAIN and E41-AGAIN plates were picked and inoculated in LB + Cm12.5; J101-31, J101-E5 and J101-E7 plasmids were extracted:

Plasmid DNA (ng/μl)
J101-31 38
J101-E5 14.5
J101-E7 14

These plasmids were digested with EcoRI and SpeI restriction enzymes and screened by gel-electrophoresis; a medium-size agarose gel was then prepared.
MGZ1 competent cells were prepared according to protocol. They were put in 22 100 μl test-tubes.
Gel electrophoresis was performed:

Medium size gel

Insert lengths were all correct so J101-31, J101-E5, J101-E7 were transformed from MiniPrepped DNA in MGZ1 competent cells.


E37-2, E38-1, E39-1, E40-2 and E42-1 were inoculated in 5 ml LB + Cm12.5 and grown ON for plasmid extraction.

August, 2nd

MGZ1 J101-31, J101-E5 and J101-E7 plates were all grown; one colony was picked from each of them and inoculated in LB + Cm12.5. Plasmids harboring E28-AGAIN-1, E28-AGAIN-2, E41-AGAIN-1, E28-AGAIN-2, E37-2, E38-1, E39-1, E40-2 and E42-1 were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:

Plasmid DNA (ng/μl)
E28AGAIN-1 21.4
E28AGAIN-2 21.0
E41AGAIN-1 33.7
E41AGAIN-2 27.7
E37-2 18.5
E38-1 26.0
E39-1 21.6
E40-2 19.9
E42-1 25.1

Digestions of E28AGAIN-1, E28AGAIN-2, E41AGAIN-1 and E42AGAIN-2 were performed for screening; a 5x mix was prepared and for each sample the volumes specified in the table below were used:

DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
5 16.5 0.5 EcoRI 0.5 PstI 2.5 25

A small-size agarose gel was prepared for gel electrophoresis.
TOP10 competent cells, harboring BBa_T9002 construct, were co-transformed with ENTERO4C5 to confer resistance to Cloramphenicol (12.5 mg/ml) and plated on LB agar + Amp + Cm 12.5 plates.
In the afternoon gel electrophoresis was performed:

Small size gel

Unfortunately insert length was correct only for E28-AGAIN-1 so we ligated again E41:

Ligation Name Vector Vector volume (μl) Insert Insert volume (μl) Buffer (μl) T4 Ligase (μl)
E41N E36 (S-P) 4 E3-1 (X-P) 4 1 1

Glycerol stocks were prepared according to the protocol for J101-31, J101-E5 and J101-E7.
E37-2, E38-1, E39-1, E40-2, E42-1 and E28-AGAIN-1 DNA was sent to BMR genomics for sequencing.
5 μl of E37-2, E38.1, E39-1, E40-2 and ENTERO4C5 were inoculated in 1 ml M9 + Cm12.5 for a preliminary test on the autoinducer inactivation enzyme A (AiiA) enzyme.

August, 3rd

T9002-ENTERO4C5 plate was grown and a colony was picked and inoculated in LB + Amp + Cm12.5; E41N ligation was transformed in 100 μl of MGZ1 competent cells and plated on a LB + Cm12.5 plate.
34 LB agar + Cm12.5 plates were prepared according to protocols.
E37-2, E38.1, E39-1, E40-2 and ENTERO4C5 cultures were diluted 1:100 in a final volume of 5 ml of M9 + Cm12.5 for the preliminary test; a 1000 x 75 ng/μl atc stock was prepared.
After 3 hours cultures were induced with 5 μl of atc (75 ng/ml final concentration); after 4 hours 2.5 μl of 2mM 3OC6-HSL (1:2000 dilution) were added (final 3OC6-HSL concentration 1μM) and the experiment started (t = 0 h).
250 μl samples were taken at t = 0 h, t = 1 h, and t = 4 h; O.D. at 600 nm was measured:

E37 E38 E39 E40 ENTERO4C5
t = 0 h 0.12 0.11 0.13 0.10 0.11
t = 1 h 0.22 0.15 0.21 0.28 0.19
t = 4 h 0.47 0.60 0.53 0.37 0.53

Then samples were centrifuged at 13.000 rpm; finally the supernatant was collected and stored at -20°C.
Glycerol stock for T9002-ENTERO4C5 was prepared.
Two 5 μl inocula of E32, E33, E34, E35, J101-E5, J101-31, J101-E6, J101-E7 and ENTERO4C5 were prepared in 1 ml of M9 + Cm12.5 for a preliminary test on TetR repressible promoter.

August, 4th

E41N plate was grown; three colonies were picked (E41N-1, E41N-2, E41N-3) and inoculated in LB + Cm12.5.
E32-1, E33-1, E34-1, E35-1, J101-E5-1, J101-31-1, J101-E6-1, J101-E7-1, E32-2, E33-2, E34-2, E35-2, J101-E5-2, J101-31-2, J101-E6-2, J101-E7-2, ENTERO4C5-1 and ENTERO4C5-2 were diluted 1:500 in a final volume of 1 ml. After 2 hours and 30 minutes inducible cultures were supplemented with anhydrotetracyclin (atc) at final concentrations of: 1 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml. Moreover uninduced cultures were supplemented with ddH2O.
ENTERO-RBS and T9002-ENTERO4C5 were inoculated in 1 ml of Cm12.5 for preliminary AiiA test (with supernatants collected on August, 3rd).

August, 5th

ENTERO-RBS and T9002-ENTERO4C5 cultures were diluted 1:500 in a final volume of 1 ml and 10 ml of M9 + Cm12.5 respectively; they were grown for six hours. Plasmid purification was performed for E41N-1, E41N-2 and E41N-3:

Plasmid DNA (ng/μl)
E41N-1 62.3
E41N-2 17.3
E41N-3 43.9

Screening digestion with EcoRI and PstI restriction enzymes was carried out:

Plasmid DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
E41N-1 2.5 19 0.5 EcoRI 0.5 Pstl 2.5 25
E41N-2 8.5 13 0.5 EcoRI 0.5 Pstl 2.5 25
E41N-2 3.5 18 0.5 EcoRI 0.5 Pstl 2.5 25

3OC6-HSL was diluted to known concentrations in order to induce T9002-ENTERO4C5 biosensor and obtain a reference curve for the measurements on E37, E38, E39, E40 and ENTERO4C5 supernatants.
A small-size agarose gel was prepared; in the afternoon gel electrophoresis was carried out:

Small size gel

Only E41N-1 showed the correct band, so we decided to keep it. E41N-2 and E41N-3 glycerol stocks were thrown.
TECAN test on E37, E38, E39, E40 and ENTERO4C5 supernatants was carried out; as supernatants were not diluted enough it was not possible to precisely measure 3OC6-HSL concentration as the biosensor (T9002-ENTERO4C5) worked in the saturation zone.