Team:UNIPV-Pavia/Calendar/August/week3

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:

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.