Team:UNIPV-Pavia/Calendar/August/week3

From 2011.igem.org

UNIPV TEAM 2011

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AUGUST: WEEK 3

August, 16th

Inoculum of T9002-ENTERO4C5 and ENTERO-RBS (1 ml of M9 + Cm12.5) to test again diluted supernatants of E37, E38, E39, E40 and ENTERO4C5.
Inoculum of E41N-1 and J101-4C5 for plasmid purification 6 ml of LB + Cm12.5.
Streak of E32-2, E33-2, J101-31, J101-E5 and ENTERO4C5 on an LB-agar plate + Cm12.5 to test and characterize PtetR promoter with different Ribosome Binding Sites (BBa_B0030, BBa_B0031).

August, 17th

E41N-1 and J101-4C5 plasmids were purified with mini Prep kit:

Plasmid DNA (ng/μl)
E41N-1 33.7
J101-4C5 25.4

T9002-ENTERO4C5 and ENTERO-RBS were diluted 1:500 in a final volume and 1 ml of 12 ml M9 + Cm12.5 respectively and grown for 6 hours.
3OC6-HSL was diluted to known concentrations in order to induce T9002-ENTERO4C5 biosensor and obtain a reference curve for the measurements on E37, E38, E39, E40 and ENTERO4C5 supernatants. Supernatants were diluted in M9 + Amp + Cm12.5 in order to obtain 1:500 and 1:200 final dilutions in the 200 μl-well of TECAN microplate.
In the afternoon supernatants were tested; this time results were more precise, even if negative control seemed to inactivate 3OC6-HSL as fast as other cultures.
1.5 μl of J101-4C5 purified DNA was transformed in 100 μl of MGZ1 competent cells.
E32, E33, J101-31, J101-E5 and ENTERO4C5 plate was grown so two colonies for each strain were picked and inoculated in 1 ml of M9 + Cm12.5 for PtetR characterization.
BBa_K300005 was inoculated in 6 ml of LB + Cm34 in order to extract pSB1C3 standard shipping vector.

August, 18th

J101-4C5 plate was grown and a colony was picked and inoculated in 750 μl LB + Cm12.5; in the late afternoon 250 μl glycerol 80% were added in order to prepare a glycerol stock of this culture.
Cultures involved in PtetR characterization were diluted 1:500 in a final volume of 1 ml M9 + Cm12.5 and grown for 2.5 hours. Then they were induced with 10 different final concentrations of anhydrotetracycline (atc) (0 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 8 ng/ml, 10 ng/ml50 ng/ml and 100 ng/ml).

Plasmid purification was performed for BBa_K300005:

Plasmid DNA (ng/μl)
BBa_K300005 205

Then the plasmid was digested with EcoRI and PstI endonucleases:
Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
BBa_K300005 Vector 4 16.5 1 EcoRl 1 Pstl 2.5 25

Gel electrophoresis was carried out:

Small size gel

After gel extraction, digested DNA was quantified:

Part DNA (ng/μl)
BBa_K300005 (E-P) 13

Digested DNA (pSB1C3) was stored at -20°C, in order to ligate the parts to send to the Registry.
Streak of E34-1, E35-2, J101-E7, J101-4C5 and ENTERO4C5 on an LB-agar plate + Cm12.5 to test and characterize PtetR promoter with different Ribosome Binding Sites (BBa_B0032, BBa_B0034).
Inoculum of E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 in 1 ml M9 + Cm12.5 to test the efficiency of AiiA enzyme.

August, 19th

E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 were diluted 1:500 in 6 ml M9 + Cm12.5. After 3 hours they were induced with 75 ng/ml atc; after 1 hour from induction 100 nM 3OC6-HSL was added (t = 0 h); this time also 3OC6-HSL in M9 + Cm12.5 without cells was measured. Supernatants were collected as on August,3rd.
250 μl samples were taken at t = 0 h, t = 1 h, t = 4 h and t = 21 h; O.D.600 and pH (which was constant at about 7) were measured:

E37 E38 E39 E40 ENTERO4C5 M9
t = 0 h 0.0068 0.0078 0.0094 0.0060 0.0077 ###
t = 2 h 0.031 0.026 0.032 0.028 0.036 ###
t = 4 h 0.105 0.096 0.109 0.098 0.112 ###
t = 21 h 0.474 0.463 0.496 0.485 0.450 ###

Supernatants were stored at -20°C.
Inoculum of E24-1, E25-2, E26-2 and E27-2 in LB + Amp for plasmid purification.
Inoculum of E34-1, E35-2, J101-E7, J101-4C5 and ENTERO4C5 (three colonies for part) in M9 + Cm12.5, for TECAN test on PtetR promoter.

August, 20th

1:500 dilution of test cultures in 1 ml (final volume).
After 2 hours and 30 minutes PtetR cultures were induced with atc (0 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 8 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml); after two hours and 30 minutes 200 μl of every culture were aliquoted in TECAN Infinite 200.
Plasmid purification for E24-1, E25-2, E26-2 and E27-2 was carried out:

Plasmid DNA (ng/μl)
E24-1 82.3
E25-2 66.4
E26-2 87.0
E27-2 79.2