Team:UNIPV-Pavia/Calendar/August/week3

From 2011.igem.org

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<div align="justify">
<a name="August.2C_16th"></a><h2> <span class="mw-headline">August, 16th</span></h2>
<a name="August.2C_16th"></a><h2> <span class="mw-headline">August, 16th</span></h2>
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Inoculum of E41N-1 and J101-4C5 for plasmid purification 6 ml of LB + Cm12.5.
Inoculum of E41N-1 and J101-4C5 for plasmid purification 6 ml of LB + Cm12.5.
<br>
<br>
-
Streak of E32, E33, J101-31, J101-E5 and ENTERO4C5 on an LB-agar plate + Cm12.5 to test and characterize PtetR promoter with different Ribosome Binding Sites (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0030">BBa_B0030</a>, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0031">BBa_B0031</a>).
+
Streak of E32-2, E33-2, J101-31, J101-E5 and ENTERO4C5 on an LB-agar plate + Cm12.5 to test and characterize PtetR promoter with different Ribosome Binding Sites (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0030">BBa_B0030</a>, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0031">BBa_B0031</a>).
</p>
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Cultures involved in PtetR characterization were diluted 1:500 in a final volume of 1 ml M9 + Cm12.5 and grown for 2.5 hours. Then they were induced with 10 different final concentrations of anhydrotetracycline (atc) (0 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 8 ng/ml, 10 ng/ml50 ng/ml and 100 ng/ml).
Cultures involved in PtetR characterization were diluted 1:500 in a final volume of 1 ml M9 + Cm12.5 and grown for 2.5 hours. Then they were induced with 10 different final concentrations of anhydrotetracycline (atc) (0 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 8 ng/ml, 10 ng/ml50 ng/ml and 100 ng/ml).
<br>
<br>
-
Plasmid purification was performed for <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K300005">BBa_K300005</a>:
+
<p>Plasmid purification was performed for <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K300005">BBa_K300005</a>:</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"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K300005">BBa_K300005</a></td>
 +
      <td class="row">205</td>
 +
  </tr>
 +
 
 +
 
 +
</table>
 +
</center>
 +
<br>
 +
Then the plasmid was digested with EcoRI and PstI endonucleases:
 +
 
 +
<center>
 +
<table class="data">
 +
    <tr>
 +
      <td class="row"><b>Plasmid</b></td>
 +
      <td class="row"><b>Kind</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"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K300005">BBa_K300005</a></td>
 +
      <td class="row">Vector</td>
 +
      <td class="row">4</td>
 +
      <td class="row">16.5</td>
 +
      <td class="row">1 EcoRl</td>
 +
      <td class="row">1 Pstl</td>
 +
      <td class="row">2.5</td>
 +
      <td class="row">25</td>
 +
  </tr>
 +
</table>
 +
</center>
 +
 
 +
<p>
 +
Gel electrophoresis was carried out:
 +
</p>
 +
 
 +
 
 +
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 550px;"><a href="/File:UNIPV_BBa_K30005_EP.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/b/b7/UNIPV_BBa_K30005_EP.PNG" class="thumbimage" height="80%" width="80%"></a>  <div class="thumbcaption">Small size gel</div></div></div></div>
 +
 
 +
<p>
 +
After gel extraction, digested DNA was quantified:
 +
</p>
 +
 
 +
<center>
 +
<table class="data">
 +
    <tr>
 +
      <td class="row"><b>Part</b></td>
 +
      <td class="row"><b>DNA (ng/&mu;l)</b></td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K300005">BBa_K300005</a> (E-P)</td>
 +
      <td class="row">13</td>
 +
  </tr>
 +
</table>
 +
</center>
 +
 
 +
 
 +
<p>
 +
Digested DNA (<a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a>) was stored at -20°C, in order to ligate the parts to send to the Registry.
 +
<br>
 +
Streak of E34-1, E35-2, J101-E7, J101-4C5 and ENTERO4C5 on an LB-agar plate + Cm12.5 to test and characterize PtetR promoter with different Ribosome Binding Sites (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0032">BBa_B0032</a>, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034">BBa_B0034</a>).
 +
<br>
 +
Inoculum of E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 in 1 ml M9 + Cm12.5 to test the efficiency of AiiA enzyme.
 +
</p>
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
<a name="August.2C_19th"></a><h2> <span class="mw-headline">August, 19th</span></h2>
 +
 +
<p>
 +
E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 were diluted 1:500 in 6 ml M9 + Cm12.5. After 3 hours they were induced with 75 ng/ml atc; after 1 hour from induction 100 nM 3OC<sub><small>6</small></sub>-HSL was added (t = 0 h); this time also 3OC<sub><small>6</small></sub>-HSL in M9 + Cm12.5 without cells was measured. Supernatants were collected as on <a href="/Team:UNIPV-Pavia/Calendar/August/week1#August.2C_3rd">August,3rd</a>.
 +
<br>
 +
250 μl samples were taken at t = 0 h, t = 1 h, t = 4 h and t = 21 h; O.D.<sub><small>600</small></sub> and pH (which was constant at about 7) were measured:
 +
</p>
 +
 +
 +
<center>
 +
<table class="data">
 +
    <tr>
 +
      <td class="row"></td>
 +
      <td class="row"><b>E37</b></td>
 +
      <td class="row"><b>E38</b></td>
 +
      <td class="row"><b>E39</b></td>
 +
      <td class="row"><b>E40</b></td>
 +
      <td class="row"><b>ENTERO4C5</b></td>
 +
      <td class="row"><b>M9</b></td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row"><b>t = 0 h</b></td>
 +
      <td class="row">0.0068</td>
 +
      <td class="row">0.0078</td>
 +
      <td class="row">0.0094</td>
 +
      <td class="row">0.0060</td>
 +
      <td class="row">0.0077</td>
 +
      <td class="row">###</td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row"><b>t = 2 h</b></td>
 +
      <td class="row">0.031</td>
 +
      <td class="row">0.026</td>
 +
      <td class="row">0.032</td>
 +
      <td class="row">0.028</td>
 +
      <td class="row">0.036</td>
 +
      <td class="row">###</td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row"><b>t = 4 h</b></td>
 +
      <td class="row">0.105</td>
 +
      <td class="row">0.096</td>
 +
      <td class="row">0.109</td>
 +
      <td class="row">0.098</td>
 +
      <td class="row">0.112</td>
 +
      <td class="row">###</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row"><b>t = 21 h</b></td>
 +
      <td class="row">0.474</td>
 +
      <td class="row">0.463</td>
 +
      <td class="row">0.496</td>
 +
      <td class="row">0.485</td>
 +
      <td class="row">0.450</td>
 +
      <td class="row">###</td>
 +
  </tr>
 +
 +
 +
</table>
 +
</center>
 +
 +
<p>
 +
Supernatants were stored at -20°C.
 +
<br>
 +
Inoculum of E24-1, E25-2, E26-2 and E27-2 in LB + Amp for plasmid purification.
 +
<br>
 +
Inoculum of E34-1, E35-2, J101-E7, J101-4C5 and ENTERO4C5 (three colonies for part) in M9 + Cm12.5, for TECAN test on PtetR promoter.
 +
</p>
 +
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
 +
<a name="August.2C_20th"></a><h2> <span class="mw-headline">August, 20th</span></h2>
 +
 +
<p>
 +
1:500 dilution of test cultures in 1 ml (final volume).
 +
<br>
 +
After 2 hours and 30 minutes PtetR cultures were induced with atc (0 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 8 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml); after two hours and 30 minutes 200 &mu;l of every culture were aliquoted in TECAN Infinite 200.
 +
<br>
 +
Plasmid purification for E24-1, E25-2, E26-2 and E27-2 was carried out:
 +
</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">E24-1</td>
 +
      <td class="row">82.3</td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row">E25-2</td>
 +
      <td class="row">66.4</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row">E26-2</td>
 +
      <td class="row">87.0</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row">E27-2</td>
 +
      <td class="row">79.2</td>
 +
  </tr>
 +
 +
</table>
 +
</center>
 +
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
<br>
<div>
<div>
<span style="float:left;">
<span style="float:left;">
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{{endcalendar}}
{{endcalendar}}

Latest revision as of 15:52, 20 September 2011

UNIPV TEAM 2011

March
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    

April
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

May
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

June
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

July
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

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
      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

October
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

AUGUST: WEEK 3

August, 16th

Inoculum of T9002-ENTERO4C5 and ENTERO-RBS (1 ml of M9 + Cm12.5) to test again diluted supernatants of E37, E38, E39, E40 and ENTERO4C5.
Inoculum of E41N-1 and J101-4C5 for plasmid purification 6 ml of LB + Cm12.5.
Streak of E32-2, E33-2, J101-31, J101-E5 and ENTERO4C5 on an LB-agar plate + Cm12.5 to test and characterize PtetR promoter with different Ribosome Binding Sites (BBa_B0030, BBa_B0031).

August, 17th

E41N-1 and J101-4C5 plasmids were purified with mini Prep kit:

Plasmid DNA (ng/μl)
E41N-1 33.7
J101-4C5 25.4

T9002-ENTERO4C5 and ENTERO-RBS were diluted 1:500 in a final volume and 1 ml of 12 ml M9 + Cm12.5 respectively and grown for 6 hours.
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. Supernatants were diluted in M9 + Amp + Cm12.5 in order to obtain 1:500 and 1:200 final dilutions in the 200 μl-well of TECAN microplate.
In the afternoon supernatants were tested; this time results were more precise, even if negative control seemed to inactivate 3OC6-HSL as fast as other cultures.
1.5 μl of J101-4C5 purified DNA was transformed in 100 μl of MGZ1 competent cells.
E32, E33, J101-31, J101-E5 and ENTERO4C5 plate was grown so two colonies for each strain were picked and inoculated in 1 ml of M9 + Cm12.5 for PtetR characterization.
BBa_K300005 was inoculated in 6 ml of LB + Cm34 in order to extract pSB1C3 standard shipping vector.

August, 18th

J101-4C5 plate was grown and a colony was picked and inoculated in 750 μl LB + Cm12.5; in the late afternoon 250 μl glycerol 80% were added in order to prepare a glycerol stock of this culture.
Cultures involved in PtetR characterization were diluted 1:500 in a final volume of 1 ml M9 + Cm12.5 and grown for 2.5 hours. Then they were induced with 10 different final concentrations of anhydrotetracycline (atc) (0 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 8 ng/ml, 10 ng/ml50 ng/ml and 100 ng/ml).

Plasmid purification was performed for BBa_K300005:

Plasmid DNA (ng/μl)
BBa_K300005 205

Then the plasmid was digested with EcoRI and PstI endonucleases:
Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
BBa_K300005 Vector 4 16.5 1 EcoRl 1 Pstl 2.5 25

Gel electrophoresis was carried out:

Small size gel

After gel extraction, digested DNA was quantified:

Part DNA (ng/μl)
BBa_K300005 (E-P) 13

Digested DNA (pSB1C3) was stored at -20°C, in order to ligate the parts to send to the Registry.
Streak of E34-1, E35-2, J101-E7, J101-4C5 and ENTERO4C5 on an LB-agar plate + Cm12.5 to test and characterize PtetR promoter with different Ribosome Binding Sites (BBa_B0032, BBa_B0034).
Inoculum of E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 in 1 ml M9 + Cm12.5 to test the efficiency of AiiA enzyme.

August, 19th

E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 were diluted 1:500 in 6 ml M9 + Cm12.5. After 3 hours they were induced with 75 ng/ml atc; after 1 hour from induction 100 nM 3OC6-HSL was added (t = 0 h); this time also 3OC6-HSL in M9 + Cm12.5 without cells was measured. Supernatants were collected as on August,3rd.
250 μl samples were taken at t = 0 h, t = 1 h, t = 4 h and t = 21 h; O.D.600 and pH (which was constant at about 7) were measured:

E37 E38 E39 E40 ENTERO4C5 M9
t = 0 h 0.0068 0.0078 0.0094 0.0060 0.0077 ###
t = 2 h 0.031 0.026 0.032 0.028 0.036 ###
t = 4 h 0.105 0.096 0.109 0.098 0.112 ###
t = 21 h 0.474 0.463 0.496 0.485 0.450 ###

Supernatants were stored at -20°C.
Inoculum of E24-1, E25-2, E26-2 and E27-2 in LB + Amp for plasmid purification.
Inoculum of E34-1, E35-2, J101-E7, J101-4C5 and ENTERO4C5 (three colonies for part) in M9 + Cm12.5, for TECAN test on PtetR promoter.

August, 20th

1:500 dilution of test cultures in 1 ml (final volume).
After 2 hours and 30 minutes PtetR cultures were induced with atc (0 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 8 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml); after two hours and 30 minutes 200 μl of every culture were aliquoted in TECAN Infinite 200.
Plasmid purification for E24-1, E25-2, E26-2 and E27-2 was carried out:

Plasmid DNA (ng/μl)
E24-1 82.3
E25-2 66.4
E26-2 87.0
E27-2 79.2