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SEPTEMBER: WEEK 2
September, 5th
T9002-ENTERO and ENTERO-4C5 were diluted 1:200 and 1:800 to perform two different tests on supernatants (collected on August, 31st, from cultures and media at different pHs, and on September, 3rd, from cultures expressing AiiA enzyme in high copy number plasmids).
September, 6th
T9002-ENTERO and ENTERO-RBS were diluted 1:200 in a final volume of 1 ml and 18 ml respectively. After two hours 190 μl were aliquoted in 96-well microplate and induced with 10 μl of supernatants and 3OC6-HSL known concentrations.
September, 7th
BMR Genomics results were ready; all the parts were correct, except for E4-1C3, so E4-2 was again inoculated.
September, 8th
T9002-ENTERO and ENTERO-RBS were diluted 1:200 in M9 + Cm12.5; after growing two hours they were aliquoted in 96-well microplate. T9002-ENTERO wells were induced with 10 μl 3OC6-HSL (1:20 dilution) to final concentrations 0 nM, 0.005 nM, 0.01 nM, 0.025 nM, 0.04 nM, 0.05 nM, 0.075 nM, 0.1 nM, 0.15 nM, 0.2 nM, 0.25 nM and 0.5 nM in order to test the sensitivity of the biosensor.
E4-2 was digested:
Digestion was kept for three hours at 37°C; a small size agarose gel was prepared and gel electrophoresis was carried out: DNA bands were cut and gel extracted, even if E4-2 (EP) showed extra-bands. E4 was then transferred in pSB1C3:
ENTERO4C5 was inoculated in 1 ml M9 + Cm12.5 at different pHs (5.9, 6.3, 6.7, 6.9 and 7.2) and in 1 ml LB + Cm12.5 (pH measured = 6.74).
September, 9th
ENTERO4C5 at different pHs cultures were diluted 1:100 and, after two hours, they were supplemented with 100 nM 3OC6-HSL; supernatants from these tubes were collected at 0 h, 1 h, 2 h and 4 h from supplementation (also M9 without cultures at differents pHs was monitored).
September, 10th
E21, E22, E23, BBa_R0040 in BBa_J61002 and E4N-1C3 plates showed colonies; three were picked for E4N-1C3 while one for the others and inoculated in LB with the proper antiobiotic.
September, 11th
T9002-ENTERO and ENTERO-RBS were diluted 1:100, grown for two hours and induced with E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 cultures at t = 2 h and t = 4 h supernatants to measure 3OC6-HSL concentration.
All the clones were digested with EcoRI and PstI endonucleases and screened by gel electrophoresis; they all showed the correct bands. E31-1, E41N-1, E42-1, E43-3 and ENTERO4C5 were inoculated in M9 + Cm12.5 pH ~= 6.
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Team:UNIPV-Pavia/Calendar/September/week2
From 2011.igem.org
(Difference between revisions)
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</h2> | </h2> | ||
<div class="cleared"></div> | <div class="cleared"></div> | ||
- | <div class="art-postcontent"> | + | <div class="art-postcontent" style="margin-right: 10pt;"> |
<p style="text-align:left;"> | <p style="text-align:left;"> | ||
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<p><a name="indice"/> | <p><a name="indice"/> | ||
</p> | </p> | ||
+ | <div align="justify"> | ||
<a name="September.2C_5th"></a><h2> <span class="mw-headline">September, 5th</span></h2> | <a name="September.2C_5th"></a><h2> <span class="mw-headline">September, 5th</span></h2> | ||
<p> | <p> | ||
Line 41: | Line 42: | ||
</p> | </p> | ||
- | <div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 550px;"><a href="/File: | + | <div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 550px;"><a href="/File:UNIPV_06_09_2011_E3Screening2.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/a/a5/UNIPV_06_09_2011_E3Screening2.PNG" class="thumbimage" height="80%" width="80%"></a> <div class="thumbcaption">Medium size gel</div></div></div></div> |
<p> | <p> | ||
Line 57: | Line 58: | ||
<a name="September.2C_7th"></a><h2> <span class="mw-headline">September, 7th</span></h2> | <a name="September.2C_7th"></a><h2> <span class="mw-headline">September, 7th</span></h2> | ||
<p> | <p> | ||
- | BMR Genomics results were ready; all the parts were correct, except for E4-1C3, so E4 was again inoculated. | + | BMR Genomics results were ready; all the parts were correct, except for E4-1C3, so E4-2 was again inoculated. |
<br> | <br> | ||
Plates showed a lot of colonies; one of them was picked for eache plate and inoculated in 1 ml LB + Amp. Also RBS32 in MGZ1 was inoculated in 1 ml LB + Amp. | Plates showed a lot of colonies; one of them was picked for eache plate and inoculated in 1 ml LB + Amp. Also RBS32 in MGZ1 was inoculated in 1 ml LB + Amp. | ||
Line 66: | Line 67: | ||
<br> | <br> | ||
T9002-ENTERO and ENTERO-RBS were again inoculated in M9 + Amp + Cm12.5 to test T9002-ENTERO sensitivity. | T9002-ENTERO and ENTERO-RBS were again inoculated in M9 + Amp + Cm12.5 to test T9002-ENTERO sensitivity. | ||
+ | <br> | ||
+ | E3O-1C3-2 and E3O-1C3-3 were plasmid purified: | ||
</p> | </p> | ||
+ | |||
+ | |||
+ | <center> | ||
+ | <table class="data"> | ||
+ | <tr> | ||
+ | <td class="row"><b>Plasmid</b></td> | ||
+ | <td class="row"><b>DNA (ng/μl)</b></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td class="row">E3O-1C3-2</td> | ||
+ | <td class="row">62.0</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td class="row">E3O-1C3-3</td> | ||
+ | <td class="row">75.6</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </center> | ||
Line 73: | Line 96: | ||
<a name="September.2C_8th"></a><h2> <span class="mw-headline">September, 8th</span></h2> | <a name="September.2C_8th"></a><h2> <span class="mw-headline">September, 8th</span></h2> | ||
+ | <p> | ||
+ | T9002-ENTERO and ENTERO-RBS were diluted 1:200 in M9 + Cm12.5; after growing two hours they were aliquoted in 96-well microplate. T9002-ENTERO wells were induced with 10 μl 3OC<small><sub>6</sub></small>-HSL (1:20 dilution) to final concentrations 0 nM, 0.005 nM, 0.01 nM, 0.025 nM, 0.04 nM, 0.05 nM, 0.075 nM, 0.1 nM, 0.15 nM, 0.2 nM, 0.25 nM and 0.5 nM in order to test the sensitivity of the biosensor. | ||
+ | <br> | ||
+ | E24, E25, E26 and E27 in MGZ1 were glycerol stocked; they were also diluted 1:100 in 4 ml M9 + Amp to test AiiA efficiency in HC number plasmids (also a negative control, RBS, and a falcon with M9 + Amp only were prepared). After growing two hours they were induced with 100 ng/ml atc and, after one hour, 3OC<small><sub>6</sub></small>-HSL was added at a final 100 nM concentration. Samples were taken at the moment of supplementation with 3OC<small><sub>6</sub></small>-HSL, after 1 hour, two hours, four hours and seven hours. | ||
+ | <br> | ||
+ | E4-2 was purified: | ||
+ | </p> | ||
+ | <center> | ||
+ | <table class="data"> | ||
+ | <tr> | ||
+ | <td class="row"><b>Plasmid</b></td> | ||
+ | <td class="row"><b>DNA (ng/μl)</b></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="row">E4-2</td> | ||
+ | <td class="row">53.6</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </center> | ||
+ | <p> | ||
+ | E4-2 was digested: | ||
+ | </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 (μl)</b></td> | ||
+ | <td class="row"><b>H<small><sub>2</sub></small>O (μl)</b></td> | ||
+ | <td class="row"><b>Enzyme 1 (μl)</b></td> | ||
+ | <td class="row"><b>Enzyme 2 (μl)</b></td> | ||
+ | <td class="row"><b>Buffer H (μl)</b></td> | ||
+ | <td class="row"><b>Final Volume (μl)</b></td> | ||
+ | </tr> | ||
- | < | + | <tr> |
+ | <td class="row">E4-2</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> | ||
+ | Digestion was kept for three hours at 37°C; a small size agarose gel was prepared and gel electrophoresis was carried out: | ||
+ | </p> | ||
+ | <div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 550px;"><a href="/File:UNIPV_08_09_2011_E4_2_EP.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7c/UNIPV_08_09_2011_E4_2_EP.PNG" class="thumbimage" height="50%" width="50%"></a> <div class="thumbcaption">Small size gel</div></div></div></div> | ||
+ | |||
+ | <p> | ||
+ | DNA bands were cut and gel extracted, even if E4-2 (EP) showed extra-bands. E4 was then transferred in pSB1C3: | ||
+ | </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 (μl)</b></td> | ||
+ | <td class="row"><b>Insert</b></td> | ||
+ | <td class="row"><b>Insert volume (μl)</b></td> | ||
+ | <td class="row"><b>Buffer (μl)</b></td> | ||
+ | <td class="row"><b>T4 Ligase (μl)</b></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td class="row"><b>E4N-1C3</b></td> | ||
+ | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td> | ||
+ | <td class="row">2</td> | ||
+ | <td class="row">E4-2 (E-P)</td> | ||
+ | <td class="row">6</td> | ||
+ | <td class="row">1</td> | ||
+ | <td class="row">1</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </center> | ||
+ | |||
+ | <p> | ||
+ | ENTERO4C5 was inoculated in 1 ml M9 + Cm12.5 at different pHs (5.9, 6.3, 6.7, 6.9 and 7.2) and in 1 ml LB + Cm12.5 (pH measured = 6.74). | ||
+ | <br> | ||
+ | T9002-ENTERO and ENTERO-RBS were again inoculated in M9 + Cm12.5 in order to test AiiA HC supernatants collected during the day. | ||
+ | </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="September. | + | <a name="September.2C_9th"></a><h2> <span class="mw-headline">September, 9th</span></h2> |
+ | <p> | ||
+ | ENTERO4C5 at different pHs cultures were diluted 1:100 and, after two hours, they were supplemented with 100 nM 3OC<sub><small>6</small></sub>-HSL; supernatants from these tubes were collected at 0 h, 1 h, 2 h and 4 h from supplementation (also M9 without cultures at differents pHs was monitored). | ||
+ | <br> | ||
+ | E21, E22, E23 and BBa_R0040 in BBa_J61002 were transferred in E. coli MGZ1 competent cells; E4N-1C3 ligation was transformed in E. coli TOP10. | ||
+ | <br> | ||
+ | Inoculum of T9002-ENTERO and ENTERO-RBS to test SN at different pHs; inoculum of E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 in M9 + Cm12.5 at pH 5.9 in order to test AiiA efficiency, varying atc induction (6 mg/ml, 8 mg/ml and 100 mg/ml). | ||
+ | </p> | ||
+ | <div align="right"><small><a href="#indice" title="">^top</a></small></div> | ||
+ | |||
+ | <a name="September.2C_10th"></a><h2> <span class="mw-headline">September, 10th</span></h2> | ||
+ | |||
+ | <p> | ||
+ | E21, E22, E23, BBa_R0040 in BBa_J61002 and E4N-1C3 plates showed colonies; three were picked for E4N-1C3 while one for the others and inoculated in LB with the proper antiobiotic. | ||
+ | <br> | ||
+ | In the morning T9002-ENTERO and ENTERO-RBS were diluted 1:100 and, after two hours, aliquoted in TECAN microplates reader; T9002-ENTERO was also induced with supernatants collected on <a href="https://2011.igem.org/Team:UNIPV-Pavia/Calendar/September/week2#September.2C_9th">September, 9th</a> (different pHs). | ||
+ | <br> | ||
+ | E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 were also diluted 1:100 in a 4 ml final volume; after two hours the were induced with different atc concentrations (6 mg/ml, 8 mg/ml and 100 mg/ml). Waiting one more hour, they were supplemented with 3OC<sub><small>6</small></sub>-HSL 100 nM. Supernatants were collected at t = 0 h, t = 1 h, t = 2 h and t = 4 h. | ||
+ | <br> | ||
+ | In the afternoon T9002-ENTERO and ENTERO-RBS were again diluted 1:100, grown for two hours and induced with E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 cultures at t = 0 h and t = 1 h supernatants to measure 3OC<sub><small>6</small></sub>-HSL concentration. | ||
+ | <br> | ||
+ | 14 LB agar + Cm12.5 plates were prepared. | ||
+ | <br> | ||
+ | Inoculum of T9002-ENTERO and ENTERO-RBS. | ||
+ | </p> | ||
Line 96: | Line 225: | ||
<a name="September.2C_11th"></a><h2> <span class="mw-headline">September, 11th</span></h2> | <a name="September.2C_11th"></a><h2> <span class="mw-headline">September, 11th</span></h2> | ||
+ | <p> | ||
+ | T9002-ENTERO and ENTERO-RBS were diluted 1:100, grown for two hours and induced with E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 cultures at t = 2 h and t = 4 h supernatants to measure 3OC<sub><small>6</small></sub>-HSL concentration. | ||
+ | <br> | ||
+ | E21, E22, E23, BBa_R0040 in BBa_J61002, E4N-1C3-1, E4N-1C3-2 and E4N-1C3-3 were purified and quantified: | ||
+ | </p> | ||
- | < | + | <center> |
+ | <table class="data"> | ||
+ | <tr> | ||
+ | <td class="row"><b>Plasmid</b></td> | ||
+ | <td class="row"><b>DNA (ng/μl)</b></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="row">E21</td> | ||
+ | <td class="row">217.8</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td class="row">E22</td> | ||
+ | <td class="row">176</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr> | ||
+ | <td class="row">E23</td> | ||
+ | <td class="row">146</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr> | ||
+ | <td class="row">BBa_R0040 in BBa_J61002</td> | ||
+ | <td class="row">82</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr> | ||
+ | <td class="row">E4N-1C3-1</td> | ||
+ | <td class="row">79.4</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr> | ||
+ | <td class="row">E4N-1C3-2</td> | ||
+ | <td class="row">77.3</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr> | ||
+ | <td class="row">E4N-1C3-3</td> | ||
+ | <td class="row">87.6</td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | </center> | ||
+ | |||
+ | <p> | ||
+ | All the clones were digested with EcoRI and PstI endonucleases and screened by gel electrophoresis; they all showed the correct bands. | ||
+ | </p> | ||
+ | |||
+ | |||
+ | <div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 550px;"><a href="/File:UNIPV_11_09_2011_E21_E22_E23_R0040_E4-1C3-1_E4-1C3-2_E4-1C3-3.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/a/a7/UNIPV_11_09_2011_E21_E22_E23_R0040_E4-1C3-1_E4-1C3-2_E4-1C3-3.PNG" class="thumbimage" height="80%" width="80%"></a> <div class="thumbcaption">Small size gel</div></div></div></div> | ||
+ | |||
+ | <p> | ||
+ | E31-1, E41N-1, E42-1, E43-3 and ENTERO4C5 were inoculated in M9 + Cm12.5 pH ~= 6. | ||
+ | </p> | ||
+ | |||
+ | <div align="right"><small><a href="#indice" title="">^top</a></small></div> | ||
+ | <br> | ||
<div> | <div> | ||
<span style="float:left;"> | <span style="float:left;"> | ||
Line 112: | Line 307: | ||
</span> | </span> | ||
</div> | </div> | ||
- | + | </div> | |
</html> | </html> | ||
{{endcalendar}} | {{endcalendar}} |
Latest revision as of 10:17, 18 September 2011