Team:UNIPV-Pavia/Calendar/August/week5

From 2011.igem.org

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<a name="August.2C_29th"></a><h2> <span class="mw-headline">August, 29th</span></h2>
<a name="August.2C_29th"></a><h2> <span class="mw-headline">August, 29th</span></h2>
<p>
<p>
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5 flasks were prepared with 29.6 ml H<sub><small>2</small></sub>O, 200 &mu;l glycerol 80 %, 8 ml M9 salts 5 x and their pH was adjusted to 5.9, 6.3, 6.7, 6.9 and 7.2; they were autoclaved in order to prepare M9 glycerol supplemented minimal medium.
5 flasks were prepared with 29.6 ml H<sub><small>2</small></sub>O, 200 &mu;l glycerol 80 %, 8 ml M9 salts 5 x and their pH was adjusted to 5.9, 6.3, 6.7, 6.9 and 7.2; they were autoclaved in order to prepare M9 glycerol supplemented minimal medium.
<br>
<br>
-
E43-3 DNA was digested with Ecori and PstI restriction nucleases in order to screen the part length by gel-electrophoresis; it showed the correct band.
+
E43-3 DNA was digested with EcorI and PstI restriction nucleases in order to screen the part length by gel-electrophoresis; it showed the correct band.
 +
</p>
 +
 
 +
 
 +
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 550px;"><a href="/File:UNIPV_29_08_2011_E43-3%28EP%29.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/10/UNIPV_29_08_2011_E43-3%28EP%29.png" class="thumbimage" height="50%" width="50%"></a>  <div class="thumbcaption">Small size gel</div></div></div></div>
 +
 
 +
<p>
 +
Plates incubated on <a name="August.2C_27th">August, 27th</a> were all grown, except for J101-31-1C3; for each of them a colony was picked and inoculated in LB + Cm34, 5 ml, while for J101-E7 two colonies were inoculated in LB + Cm34, 5 ml.
 +
<br>
 +
E43-2 was again inoculated in order to screen the insert length with gel electrophoresis.
</p>
</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_30th"></a><h2> <span class="mw-headline">August, 30th</span></h2>
 +
<p>
 +
Glycerol stocks were prepared for E2-1C3, E3-1C3, E4-1C3, E5-1C3, E6-1C3, E7-1C3, E9-1C3, E10-1C3, E11-1C3, J101-E5-1C3, J101-E7-1C3-1 and J101-E7-1C3-2.
 +
<br>
 +
DNA of each culture was purified:
 +
</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">E2-1C3</td>
 +
      <td class="row">90.2</td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row">E3-1C3</td>
 +
      <td class="row">103.7</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row">E4-1C3</td>
 +
      <td class="row">72.4</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row">E5-1C3</td>
 +
      <td class="row">78.0</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row">E6-1C3</td>
 +
      <td class="row">79.1</td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row">E7-1C3</td>
 +
      <td class="row">77.8</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row">E9-1C3</td>
 +
      <td class="row">73.5</td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row">E10-1C3</td>
 +
      <td class="row">161.8</td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row">E11-1C3</td>
 +
      <td class="row">68.8</td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row">J101-E5-1C3</td>
 +
      <td class="row">216.2</td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row">J101-E7-1C3-1</td>
 +
      <td class="row">74.7</td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row">J101-E7-1C3-2</td>
 +
      <td class="row">69.2</td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row">E43-2</td>
 +
      <td class="row">16.6</td>
 +
  </tr>
 +
 +
</table>
 +
</center>
 +
 +
<p>
 +
Each sample was digested with EcoRI and PstI endonucleases (a mix 13 x was prepared for high copy number plasmid parts):
 +
</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>EcoRI (&mu;l)</b></td>
 +
      <td class="row"><b>PstI (&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">2</td>
 +
      <td class="row">19.5</td>
 +
      <td class="row">0.5</td>
 +
      <td class="row">0.5</td>
 +
      <td class="row">2.5</td>
 +
      <td class="row">25</td>
 +
  </tr>
 +
</table>
 +
</center>
 +
 +
<p>
 +
while for E43-2 10 &mu;l were digested. In the afternoon the parts were screened by gel electrophoresis:
 +
</p>
 +
 +
 +
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 550px;"><a href="/File:UNIPV_30_08_2011_ScreeningTrasferimenti_E43_2_EP.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/2/25/UNIPV_30_08_2011_ScreeningTrasferimenti_E43_2_EP.png" class="thumbimage" height="80%" width="80%"></a>  <div class="thumbcaption">Medium size gel</div></div></div></div>
 +
 +
<p>
 +
All parts showed the correct insert length except for E3-1C3. We decided to pick and inoculate another colony from the plate, named E3-1C3-2.
 +
<br>
 +
40 &mu;l CaCl<sub><small>2</small></sub> 1M, 800 &mu;l Casamino Acids 10 %, 80 &mu;l MgSO<sub><small>4</small></sub> 1M and 1.36 ml thiamine were added to previously autoclaved flasks to prepare M9 at different pHs.
 +
<br>
 +
Inoculum of BBa_R0040 in J61002, E3-1 in 5 ml LB + Amp and J101-31 in 8 ml LB + Cm12.5.
 +
<br>
 +
Inoculum of ENTERO-4C5 in different pH M9 to test 3OC<small><sub>6</sub></small>-HSL degradation.
 +
</p>
 +
 +
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
<a name="August.2C_31st"></a><h2> <span class="mw-headline">August, 31st</span></h2>
 +
<p>
 +
E2-1C3, E4-1C3, E5-1C3, E6-1C3, E7-1C3, E9-1C3, E10-1C3, E11-1C3, J101-E5-1C3, J101-E7-1C3-1, E43-2 and E43-3 DNA samples were prepared and sent to BMR Genomics for sequencing.
 +
<br>
 +
ENTERO-4C5 cutlures were diulted 1:200 in a final volume of 5 ml M9. After 2 hours 3OC<small><sub>6</sub></small>-HSL was added to a final concentration of 100 nM and the first supernatant sample was collected. After 1 hour, 4 hours and 22 hours from 3OC<small><sub>6</sub></small>-HSL supplementation samples were again collected; supernatants were all stored at -20°C in order to measure 3OC<small><sub>6</sub></small>-HSL concentration with <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.
 +
<br>
 +
BBa_R0040 in J61002, E3-1 and J101-31 were extracted with MiniPrep kit; here are their quantifications:
 +
</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">BBa_R0040 in J61002</td>
 +
      <td class="row">79.1</td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row">E3-1</td>
 +
      <td class="row">63.7</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row">J101-31</td>
 +
      <td class="row">9.9</td>
 +
  </tr>
 +
</table>
 +
</center>
 +
 +
<p>
 +
These plasmids were digested with EcoRI and PstI endonucleases to transfer them in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>:
 +
</p>
 +
 +
<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">BBa_R0040 in J61002</td>
 +
      <td class="row">Insert</td>
 +
      <td class="row">12.5</td>
 +
      <td class="row">8</td>
 +
      <td class="row">1 EcoRI</td>
 +
      <td class="row">1 PstI</td>
 +
      <td class="row">2.5</td>
 +
      <td class="row">25</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row">E3-1</td>
 +
      <td class="row">Insert</td>
 +
      <td class="row">15.5</td>
 +
      <td class="row">5</td>
 +
      <td class="row">1 EcoRI</td>
 +
      <td class="row">1 PstI</td>
 +
      <td class="row">2.5</td>
 +
      <td class="row">25</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row"J101-31</td>
 +
      <td class="row">Insert</td>
 +
      <td class="row">20.5</td>
 +
      <td class="row">0</td>
 +
      <td class="row">1 EcoRI</td>
 +
      <td class="row">1 PstI</td>
 +
      <td class="row">2.5</td>
 +
      <td class="row">25</td>
 +
  </tr>
 +
</table>
 +
</center>
 +
 +
<p>
 +
In gel electrophoresis all inserts showed the correct length:
 +
</p>
 +
 +
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 550px;"><a href="/File:UNIPV_31_08_2011_R0040_E3-1_J10131_EP.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/5d/UNIPV_31_08_2011_R0040_E3-1_J10131_EP.png" 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">BBa_R0040 in -j61002 (E-P)</td>
 +
      <td class="row">7.9</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row">E3-1 (E-P)</td>
 +
      <td class="row">2.8</td>
 +
  </tr>
 +
 +
 +
  <tr>
 +
      <td class="row">J101-31 (E-P)</td>
 +
      <td class="row">1.5</td>
 +
  </tr>
 +
</table>
 +
</center>
 +
 +
<p>
 +
Then ligations were performed in a final volume of 10 &mu;l:
 +
</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>BBa_R0040 in J61002-1C3</b></td>
 +
      <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td>     
 +
      <td class="row">1.5</td>
 +
      <td class="row">BBa_R0040 in J61002 (E-P)</td>
 +
      <td class="row">6.5</td>
 +
      <td class="row">1</td>
 +
      <td class="row">1</td>
 +
  </tr>
 +
 +
 +
    <tr>
 +
      <td class="row"><b>E3N-1C3</b></td>
 +
      <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td>     
 +
      <td class="row">1</td>
 +
      <td class="row">E3-1 (E-P)</td>
 +
      <td class="row">7</td>
 +
      <td class="row">1</td>
 +
      <td class="row">1</td>
 +
  </tr>
 +
 +
 +
    <tr>
 +
      <td class="row"><b>J101-31N-1C3</b></td>
 +
      <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td>     
 +
      <td class="row">1</td>
 +
      <td class="row">J101-31 (E-P)</td>
 +
      <td class="row">7</td>
 +
      <td class="row">1</td>
 +
      <td class="row">1</td>
 +
  </tr>
 +
</table>
 +
</center>
 +
 +
Glycerol stock was prepared for E3-1C3-2 and the culture was pelletted.
 +
 +
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
 +
<br>
<div>
<div>
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{{endcalendar}}
{{endcalendar}}

Latest revision as of 10:12, 18 September 2011

UNIPV TEAM 2011

March
M T W T F S S
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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
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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
      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 5

August, 29th

T9002-ENTERo and ENTERO-RBS were diluted 1:500 in M9 + Amp + Cm12.5 13 ml and 1 ml volume respectively. After six hour growth they were aliquoted in 96-well microplate (190 μl cultures) with 10 μl of supernatants collected on August, 27th and 10 μl of 3OC6-HSL.
5 flasks were prepared with 29.6 ml H2O, 200 μl glycerol 80 %, 8 ml M9 salts 5 x and their pH was adjusted to 5.9, 6.3, 6.7, 6.9 and 7.2; they were autoclaved in order to prepare M9 glycerol supplemented minimal medium.
E43-3 DNA was digested with EcorI and PstI restriction nucleases in order to screen the part length by gel-electrophoresis; it showed the correct band.

Small size gel

Plates incubated on August, 27th were all grown, except for J101-31-1C3; for each of them a colony was picked and inoculated in LB + Cm34, 5 ml, while for J101-E7 two colonies were inoculated in LB + Cm34, 5 ml.
E43-2 was again inoculated in order to screen the insert length with gel electrophoresis.

August, 30th

Glycerol stocks were prepared for E2-1C3, E3-1C3, E4-1C3, E5-1C3, E6-1C3, E7-1C3, E9-1C3, E10-1C3, E11-1C3, J101-E5-1C3, J101-E7-1C3-1 and J101-E7-1C3-2.
DNA of each culture was purified:

Plasmid DNA (ng/μl)
E2-1C3 90.2
E3-1C3 103.7
E4-1C3 72.4
E5-1C3 78.0
E6-1C3 79.1
E7-1C3 77.8
E9-1C3 73.5
E10-1C3 161.8
E11-1C3 68.8
J101-E5-1C3 216.2
J101-E7-1C3-1 74.7
J101-E7-1C3-2 69.2
E43-2 16.6

Each sample was digested with EcoRI and PstI endonucleases (a mix 13 x was prepared for high copy number plasmid parts):

DNA (μl) H2O (μl) EcoRI (μl) PstI (μl) Buffer H (μl) Final Volume (μl)
2 19.5 0.5 0.5 2.5 25

while for E43-2 10 μl were digested. In the afternoon the parts were screened by gel electrophoresis:

Medium size gel

All parts showed the correct insert length except for E3-1C3. We decided to pick and inoculate another colony from the plate, named E3-1C3-2.
40 μl CaCl2 1M, 800 μl Casamino Acids 10 %, 80 μl MgSO4 1M and 1.36 ml thiamine were added to previously autoclaved flasks to prepare M9 at different pHs.
Inoculum of BBa_R0040 in J61002, E3-1 in 5 ml LB + Amp and J101-31 in 8 ml LB + Cm12.5.
Inoculum of ENTERO-4C5 in different pH M9 to test 3OC6-HSL degradation.

August, 31st

E2-1C3, E4-1C3, E5-1C3, E6-1C3, E7-1C3, E9-1C3, E10-1C3, E11-1C3, J101-E5-1C3, J101-E7-1C3-1, E43-2 and E43-3 DNA samples were prepared and sent to BMR Genomics for sequencing.
ENTERO-4C5 cutlures were diulted 1:200 in a final volume of 5 ml M9. After 2 hours 3OC6-HSL was added to a final concentration of 100 nM and the first supernatant sample was collected. After 1 hour, 4 hours and 22 hours from 3OC6-HSL supplementation samples were again collected; supernatants were all stored at -20°C in order to measure 3OC6-HSL concentration with BBa_T9002 biosensor.
BBa_R0040 in J61002, E3-1 and J101-31 were extracted with MiniPrep kit; here are their quantifications:

Plasmid DNA (ng/μl)
BBa_R0040 in J61002 79.1
E3-1 63.7
J101-31 9.9

These plasmids were digested with EcoRI and PstI endonucleases to transfer them in pSB1C3:

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
BBa_R0040 in J61002 Insert 12.5 8 1 EcoRI 1 PstI 2.5 25
E3-1 Insert 15.5 5 1 EcoRI 1 PstI 2.5 25
Insert 20.5 0 1 EcoRI 1 PstI 2.5 25

In gel electrophoresis all inserts showed the correct length:

Small size gel

After gel-extraction digested DNA was quantified:

Part DNA (ng/μl)
BBa_R0040 in -j61002 (E-P) 7.9
E3-1 (E-P) 2.8
J101-31 (E-P) 1.5

Then ligations were performed in a final volume of 10 μl:

Ligation Name Vector Vector volume (μl) Insert Insert volume (μl) Buffer (μl) T4 Ligase (μl)
BBa_R0040 in J61002-1C3 pSB1C3 (E-P) 1.5 BBa_R0040 in J61002 (E-P) 6.5 1 1
E3N-1C3 pSB1C3 (E-P) 1 E3-1 (E-P) 7 1 1
J101-31N-1C3 pSB1C3 (E-P) 1 J101-31 (E-P) 7 1 1
Glycerol stock was prepared for E3-1C3-2 and the culture was pelletted.