Team:UNIPV-Pavia/Calendar/September/week2

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).
Samples of dry DNA were prepared for R0040 in J61002-1C3-1 and J101-31-1C3-1 and sent to BMR genomics for sequencing.
E31-1, E41N-1, E42-1, E43-2, E43-3 were washed in 1 ml of M9 + Cm12.5 in order to remove 3OC₆-HSL due to PtetR leakage; then they were diluted 1:100 in 4 ml volume (falcon tube). After two hours they were induced with a final concentration of 100 ng/ml atc. Supernatants were collected at the moment of induction, named t = 0 h and then at t = 2 h, t = 4 h and t = 6 h (also from two negative controls, ENTERO4C5 and M9).
E3N-1C3 was again transformed in 200 μl of TOP10 competent cells.
T9002-ENTERO and ENTERO-RBS were again inoculated to test the supernatants collected from E31-1, E41N-1, E42-1, E43-2, E43-3, ENTERO4C5 and M9.
E37-,2 E38-1, E39-1, E40-2 and ENTERO4C5 were inoculated in 1 ml of M9 + Cm12.5 ath pH 5.9 in order to test AiiA efficiency.
In the evening we met to plan the work of the last weeks!

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.
E37-,2 E38-1, E39-1, E40-2 and ENTERO4C5 were diluted 1:200 in a final volume of 4 ml M9 + Cm12.5 at pH 5.9. After two hours they were induced with 100 ng/ml atc and after one hour 3OC₆-HSL was added at a final concentration of 100 nM. At this time the first sample was collected from each tube; samples were also collected after three hours, five hours and twenty-four hours.
E3N-1C3 plate was grown; eight colonies from this plate were picked while three colonies were picked from a older plate. All of these colonies were PCR-screened and grown in LB + Cm34. The electrophoresis showed that only two colonies, picked from the oldest plate, had the correct length part (E3O-1C3-2 and E3O-1C3-3).

Medium size gel

17 plates of LB + Amp were prepared.
E24-2, E25-1, E26-2 and E27-2 were transformed in 100 μl of MGZ1 competent cells and plated on LB agar plates + Amp.
T9002-ENTERO and ENTERO-RBS were inoculated to test the supernatants collected today from cultures producing AiiA enzyme (in LC number plasmid pSB4C5).

September, 7th

BMR Genomics results were ready; all the parts were correct, except for E4-1C3, so E4-2 was again inoculated.
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.
T9002-ENTERO and ENTERO-RBS were diluted 1:200 and grown for two hours after which they were aliquoted in 96-well TECAN microplate to test the supernatants of AiiA in LC plasmids, collected on September, 6th.
Glycerol stocks were prepared for E3O-1C3-2 and E3O-1C3-3, according to protocols.
T9002-ENTERO and ENTERO-RBS were again inoculated in M9 + Amp + Cm12.5 to test T9002-ENTERO sensitivity.
E3O-1C3-2 and E3O-1C3-3 were plasmid purified:

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.
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, 3OC₆-HSL was added at a final 100 nM concentration. Samples were taken at the moment of supplementation with 3OC₆-HSL, after 1 hour, two hours, four hours and seven hours.
E4-2 was purified:

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:

Small size gel

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).
T9002-ENTERO and ENTERO-RBS were again inoculated in M9 + Cm12.5 in order to test AiiA HC supernatants collected during the day.

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).
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.
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).

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.
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 September, 9th (different pHs).
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 3OC₆-HSL 100 nM. Supernatants were collected at t = 0 h, t = 1 h, t = 2 h and t = 4 h.
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 3OC₆-HSL concentration.
14 LB agar + Cm12.5 plates were prepared.
Inoculum of T9002-ENTERO and ENTERO-RBS.

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.
E21, E22, E23, BBa_R0040 in BBa_J61002, E4N-1C3-1, E4N-1C3-2 and E4N-1C3-3 were purified and quantified:

All the clones were digested with EcoRI and PstI endonucleases and screened by gel electrophoresis; they all showed the correct bands.

Small size gel

E31-1, E41N-1, E42-1, E43-3 and ENTERO4C5 were inoculated in M9 + Cm12.5 pH ~= 6.