Team:UNIPV-Pavia/Calendar/August/week3

From 2011.igem.org

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<br>
Plasmid purification was performed for <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K300005">BBa_K300005</a>:
Plasmid purification was performed for <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K300005">BBa_K300005</a>:
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 +
<center>
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<table class="data">
 +
    <tr>
 +
      <td class="row"><b>Plasmid</b></td>
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      <td class="row"><b>DNA (ng/&mu;l)</b></td>
 +
  </tr>
 +
 +
  <tr>
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      <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K300005">BBa_K300005</a></td>
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      <td class="row">205</td>
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  </tr>
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 +
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</table>
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</center>
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 +
Then the plasmid was digested with EcoRI and PstI endonucleases:
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 +
<center>
 +
<table class="data">
 +
    <tr>
 +
      <td class="row"><b>Plasmid</b></td>
 +
      <td class="row"><b>Kind</b></td>
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      <td class="row"><b>DNA (&mu;l)</b></td>
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      <td class="row"><b>H<small><sub>2</sub></small>O (&mu;l)</b></td>
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      <td class="row"><b>Enzyme 1 (&mu;l)</b></td>
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      <td class="row"><b>Enzyme 2 (&mu;l)</b></td>
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      <td class="row"><b>Buffer H (&mu;l)</b></td>
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      <td class="row"><b>Final Volume (&mu;l)</b></td>
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  </tr>
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 +
  <tr>
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      <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K300005">BBa_K300005</a></td>
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      <td class="row">Vector</td>
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      <td class="row">4</td>
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      <td class="row">16.5</td>
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      <td class="row">1 EcoRl</td>
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      <td class="row">1 Pstl</td>
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      <td class="row">2.5</td>
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      <td class="row">25</td>
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  </tr>
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</table>
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</center>
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<p>
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Gel electrophoresis was carried out:
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</p>
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<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>
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      <td class="row">13</td>
 +
  </tr>
 +
</table>
 +
</center>
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 +
 +
<p>
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Digested DNA was stored at -20°C, in order to ligate the parts to send to the Registry.
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
<div align="right"><small><a href="#indice" title="">^top</a></small></div>

Revision as of 09:27, 26 August 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, 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 (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 was stored at -20°C, in order to ligate the parts to send to the Registry.