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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
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<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> | ||
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<td class="row"><b>E4N-1C3</b></td> | <td class="row"><b>E4N-1C3</b></td> | ||
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<td class="row">2</td> | <td class="row">2</td> | ||
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{{endcalendar}} | {{endcalendar}} |
Latest revision as of 10:17, 18 September 2011