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AUGUST: WEEK 4
August, 22nd
Streak of E17-2, E18-2, J101-31, J101-E5 and ENTERO-4C5 on LB agar + Cm12.5 plate to test Plux promoter with different RBSs.
August, 23rd
T9002-ENTERO and ENTERO-RBS were diluted 1:500 in a final volume of 13 ml and 1 ml respectively of M9 + Amp + Cm12.5.
August, 24th
E43 ligation was transformed in 100 μl of MGZ1 competent cells.
August, 25th
Three colonies were picked from E43 plate.
Every plasmid was digested with EcoRI and PstI resctriction endonucleases. A medium size and a small size agarose-gel were prepared and, after three hours digestion, gel-electrophoresis was carried out: As showed in images E36 did not show the correct insert band while J101-31, J101-E5 and J101-E7 showed light intensity bands as they were in a low copy number plasmid. The correct parts were gel-extracted and DNA was quantified:
BBa_R0040 in J61002 was inoculated in 5 ml LB + Amp while J101-31, J101-E5 and J101-E7 were again inoculated in 8 ml of LB + Cm12.5 for plasmid purification.
August, 26th
Glycerol stock for E43-3 was prepared according to protocols.
At this point, probably E43-2 and E43-3 tubes were confused! Anyway this plasmid were digested:
A medium size agarose-gel was prepared. BBa_R0040 in J61002 resulted incorrect, while other bands had a light intensity but we decided however to gel-extract and ligate them:
E24-2, E25-1, E26-2, E27-2 and RBS32 were inoculated in 1 ml of M9 + Amp to test AiiA efficiency in high copy number plasmids, taking cultures supernatants with a protocol similar to the one used for low copy number vectors. August, 27th
E24-2, E25-1, E26-2, E27-2 and RBS32 were diluted 1:500 in a final volume of 6 ml of M9 + Amp; a tube with 6 ml of M9 + Amp medium was also prepared. These six tubes were grown for 2 hours; then 3OC6-HSL was added at a final concentration of 100 nM. Supernatants were taken at the moment of supplementation (t = 0 h), after 1 hour, after 2 hours and after 4 hours; then they were stored at -20°C.
August, 28thInoculum of T9002-ENTERO and ENTERO-RBS in 1 ml of M9 + Amp + Cm12.5 to test AiiA efficiency in high copy number plasmids with the suprnatants collected on August, 27th.
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Team:UNIPV-Pavia/Calendar/August/week4
From 2011.igem.org
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250 ml of M9 glycerol supplemented minimal medium were prepared. | 250 ml of M9 glycerol supplemented minimal medium were prepared. | ||
<br> | <br> | ||
- | Inoculum of T9002-ENTERO and ENTERO-RBS to test supernatants collected on <a | + | Inoculum of T9002-ENTERO and ENTERO-RBS to test supernatants collected on <a href="https://2011.igem.org/Team:UNIPV-Pavia/Calendar/August/week3#August.2C_20th">August, 20th</a> to test AiiA enzyme efficiency. |
</p> | </p> | ||
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Glycerol stocks were prepared for E43-1 and E43-2 while E43-3 was grown ON; these tubes were refilled with LB + Cm12.5 in order to have a final volume of 8 ml, to increase plasmid purification efficiency. | Glycerol stocks were prepared for E43-1 and E43-2 while E43-3 was grown ON; these tubes were refilled with LB + Cm12.5 in order to have a final volume of 8 ml, to increase plasmid purification efficiency. | ||
<br> | <br> | ||
- | T9002-ENTERO and ENTERO-RBS were inoculated in 1 ml of M9 + Amp + Cm12.5 to test teh supernatants collected on <a | + | T9002-ENTERO and ENTERO-RBS were inoculated in 1 ml of M9 + Amp + Cm12.5 to test teh supernatants collected on <a href="https://2011.igem.org/Team:UNIPV-Pavia/Calendar/August/week3#August.2C_20th">August, 20th</a> and test AiiA enzyme efficiency. |
</p> | </p> | ||
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<a name="August.2C_28th"></a><h2> <span class="mw-headline">August, 28th</span></h2> | <a name="August.2C_28th"></a><h2> <span class="mw-headline">August, 28th</span></h2> | ||
<p> | <p> | ||
- | Inoculum of T9002-ENTERO and ENTERO-RBS in 1 ml of M9 + Amp + Cm12.5 to test AiiA efficiency in high copy number plasmids with the suprnatants collected on <a | + | Inoculum of T9002-ENTERO and ENTERO-RBS in 1 ml of M9 + Amp + Cm12.5 to test AiiA efficiency in high copy number plasmids with the suprnatants collected on <a href="https://2011.igem.org/Team:UNIPV-Pavia/Calendar/August/week4#August.2C_27th">August, 27th</a>. |
Revision as of 10:58, 4 September 2011