Team:UNIPV-Pavia/Calendar/September/week1

From 2011.igem.org

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<a name="September.2C_2nd"></a><h2> <span class="mw-headline">September, 2nd</span></h2>
<a name="September.2C_2nd"></a><h2> <span class="mw-headline">September, 2nd</span></h2>
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Inoculum of E24-2, E25-1, E26-2, E27-2 and RBS32 in M9 + Amp to collect supernatant after 3OC<small><sub>6</sub></small>-HSL supplementation.
Inoculum of E24-2, E25-1, E26-2, E27-2 and RBS32 in M9 + Amp to collect supernatant after 3OC<small><sub>6</sub></small>-HSL supplementation.
<br>
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Inoculum in M9 + Cm12.5 of T9002-ENTERO and ENTERO-RBS to measure 3OC<small><sub>6</sub></small>-HSL concentration in supernatants at different pHs collected on <a name="https://2011.igem.org/Team:UNIPV-Pavia/Calendar/August/week5#August.2C_31st">August, 31st</a>.
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Inoculum in M9 + Cm12.5 of T9002-ENTERO and ENTERO-RBS to measure 3OC<small><sub>6</sub></small>-HSL concentration in supernatants at different pHs collected on <a href="https://2011.igem.org/Team:UNIPV-Pavia/Calendar/August/week5#August.2C_31st">August, 31st</a>.
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 550px;"><a href="/File:UNIPV_03_09_2011_E3-1C3_2_EP.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/2/20/UNIPV_03_09_2011_E3-1C3_2_EP.PNG" class="thumbimage" height="50%" width="50%"></a>  <div class="thumbcaption">Small size gel</div></div></div></div>
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The gel did not show the correct band.
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The sample did not show the correct band.
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500 ml M9 and 1 l LB were prepared.
500 ml M9 and 1 l LB were prepared.
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<div align="right"><small><a href="#indice" title="">^top</a></small></div>
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
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<a name="September.2C_4th"></a><h2> <span class="mw-headline">September, 4th</span></h2>
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R0040 in J61002-1C3-1, R0040 in J61002-1C3-2, J101-31-1C3-1 and J101-31-1C3-2 were glycerol stocked and then plasmidic DNA was purified with MiniPrep commercial kit:
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<table class="data">
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      <td class="row"><b>Plasmid</b></td>
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      <td class="row"><b>DNA (ng/&mu;l)</b></td>
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  <tr>
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      <td class="row">R0040 in J61002-1C3-1</td>
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      <td class="row">66.2</td>
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  </tr>
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  <tr>
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      <td class="row">R0040 in J61002-1C3-2</td>
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      <td class="row">81.4</td>
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  <tr>
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      <td class="row">J101-31-1C3-1</td>
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      <td class="row">86.9</td>
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      <td class="row">J101-31-1C3-2</td>
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      <td class="row">98.3</td>
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2.5 &mu;l of each sample were digested with 0.5 &mu;l EcoRI and PstI resctriction endonucleases in a 25 &mu;l final volume. A small size agarose gel was prepared. In the afternoon electrophoresis was carried out; all samples showed the correct insert.
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 550px;"><a href="/File:UNIPV_04_09_2011_R1_R2_J10131_1_J10131_2_EP.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f4/UNIPV_04_09_2011_R1_R2_J10131_1_J10131_2_EP.PNG" class="thumbimage" height="50%" width="50%"></a>  <div class="thumbcaption">Small size gel</div></div></div></div>
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<p>
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Inoculum of T9002-ENTERO and ENTERO-RBS to measure 3OC<small><sub>6</sub></small>-HSL concentration in supernatants collected on <a href="https://2011.igem.org/Team:UNIPV-Pavia/Calendar/August/week5#August.2C_31st">August, 31st</a>.
 +
<br>
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Inoculum of E31-1, E41N-1, E42-1, E43-2, E43-3 and ENTERO-4C5 to collect the supernatants and measure the 3OC<small><sub>6</sub></small>-HSL produced by luxI enzyme.
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</p>
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<div align="right"><small><a href="#indice" title="">^top</a></small></div>
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{{endcalendar}}
{{endcalendar}}

Latest revision as of 10:16, 18 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

SEPTEMBER: WEEK 1

September, 2nd

E3-1C3-2 was purified in order to screen the part length.

Plasmid DNA (ng/μl)
E2-1C3-2 82.1

R0040 in J61002-1C3, E3N-1C3 and J101-31N-1C3 were transformed in 200 μl of TOP10 competent cells.
Inoculum of E24-2, E25-1, E26-2, E27-2 and RBS32 in M9 + Amp to collect supernatant after 3OC6-HSL supplementation.
Inoculum in M9 + Cm12.5 of T9002-ENTERO and ENTERO-RBS to measure 3OC6-HSL concentration in supernatants at different pHs collected on August, 31st.

September, 3rd

All the cultures were diluted 1:200 in M9 with the proper antibiotic. After two hours T9002-ENTERO and ENTERO-RBS were aliquoted in 96-well microplate and induced with known concentrations of 3OC6-HSL and with the supernatants collected on August, 31st.
E24-2, E25-1, E26-2, E27-2, RBS32 and sterile M9 were supplemented with 100 nM 3OC6-HSL. Supernatants were collected at t = 0 h, t = 7 h and t = 24 h from all of these tubes.
R0040 in J61002-1C3 and J101-31N-1C3 showed colonies while E3N-1C3 did not grow; two colonies were picked for each plate and inoculated in Lb + Cm34, 5 ml.
2 μl of the purified plasmidic DNA of E3-1C3-2 were digested with 0.5 μl EcoRI and PstI restriction enzymes in a 25 μl final volume; a small size agarose gel was prepared.

Small size gel

The sample did not show the correct band.
500 ml M9 and 1 l LB were prepared.

September, 4th

R0040 in J61002-1C3-1, R0040 in J61002-1C3-2, J101-31-1C3-1 and J101-31-1C3-2 were glycerol stocked and then plasmidic DNA was purified with MiniPrep commercial kit:

Plasmid DNA (ng/μl)
R0040 in J61002-1C3-1 66.2
R0040 in J61002-1C3-2 81.4
J101-31-1C3-1 86.9
J101-31-1C3-2 98.3

2.5 μl of each sample were digested with 0.5 μl EcoRI and PstI resctriction endonucleases in a 25 μl final volume. A small size agarose gel was prepared. In the afternoon electrophoresis was carried out; all samples showed the correct insert.

Small size gel

Inoculum of T9002-ENTERO and ENTERO-RBS to measure 3OC6-HSL concentration in supernatants collected on August, 31st.
Inoculum of E31-1, E41N-1, E42-1, E43-2, E43-3 and ENTERO-4C5 to collect the supernatants and measure the 3OC6-HSL produced by luxI enzyme.


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