Team:UNIPV-Pavia/Calendar/August/week1

August, 1st

Two colonies for E28-AGAIN and E41-AGAIN plates were picked and inoculated in LB + Cm12.5; J101-31, J101-E5 and J101-E7 plasmids were extracted:

Plasmid	DNA (ng/μl)
J101-31	38
J101-E5	14.5
J101-E7	14

These plasmids were digested with EcoRI and SpeI restriction enzymes and screened by gel-electrophoresis; a medium-size agarose gel was then prepared.
MGZ1 competent cells were prepared according to protocol. They were put in 22 100 μl test-tubes.
Gel electrophoresis was performed:

Insert lengths were all correct so J101-31, J101-E5, J101-E7 were transformed from MiniPrepped DNA in MGZ1 competent cells.

E37-2, E38-1, E39-1, E40-2 and E42-1 were inoculated in 5 ml LB + Cm12.5 and grown ON for plasmid extraction.

August, 2nd

MGZ1 J101-31, J101-E5 and J101-E7 plates were all grown; one colony was picked from each of them and inoculated in LB + Cm12.5. Plasmids harboring E28-AGAIN-1, E28-AGAIN-2, E41-AGAIN-1, E28-AGAIN-2, E37-2, E38-1, E39-1, E40-2 and E42-1 were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:

Plasmid	DNA (ng/μl)
E28AGAIN-1	21.4
E28AGAIN-2	21.0
E41AGAIN-1	33.7
E41AGAIN-2	27.7
E37-2	18.5
E38-1	26.0
E39-1	21.6
E40-2	19.9
E42-1	25.1

Digestions of E28AGAIN-1, E28AGAIN-2, E41AGAIN-1 and E42AGAIN-2 were performed for screening; a 5x mix was prepared and for each sample the volumes specified in the table below were used:

DNA (μl)	H2O (μl)	Enzyme 1 (μl)	Enzyme 2 (μl)	Buffer H (μl)	Final Volume (μl)
5	16.5	0.5 EcoRI	0.5 PstI	2.5	25

A small-size agarose gel was prepared for gel electrophoresis.
TOP10 competent cells, harboring BBa_T9002 construct, were co-transformed with ENTERO4C5 to confer resistance to Cloramphenicol (12.5 mg/ml) and plated on LB agar + Amp + Cm 12.5 plates.
In the afternoon gel electrophoresis was performed:

Unfortunately insert length was correct only for E28-AGAIN-1 so we ligated again E41:

Ligation Name	Vector	Vector volume (μl)	Insert	Insert volume (μl)	Buffer (μl)	T4 Ligase (μl)
E41N	E36 (S-P)	4	E3-1 (X-P)	4	1	1

Glycerol stocks were prepared according to the protocol for J101-31, J101-E5 and J101-E7.
E37-2, E38-1, E39-1, E40-2, E42-1 and E28-AGAIN-1 DNA was sent to BMR genomics for sequencing.
5 μl of E37-2, E38.1, E39-1, E40-2 and ENTERO4C5 were inoculated in 1 ml M9 + Cm12.5 for a preliminary test on the autoinducer inactivation enzyme A (AiiA) enzyme.

August, 3rd

T9002-ENTERO4C5 plate was grown and a colony was picked and inoculated in LB + Amp + Cm12.5; E41N ligation was transformed in 100 μl of MGZ1 competent cells and plated on a LB + Cm12.5 plate.
34 LB agar + Cm12.5 plates were prepared according to protocols.
E37-2, E38.1, E39-1, E40-2 and ENTERO4C5 cultures were diluted 1:100 in a final volume of 5 ml of M9 + Cm12.5 for the preliminary test; a 1000 x 75 ng/μl atc stock was prepared.
After 3 hours cultures were induced with 5 μl of atc (75 ng/ml final concentration); after 4 hours 2.5 μl of 2mM 3OC₆-HSL (1:2000 dilution) were added (final 3OC₆-HSL concentration 1μM) and the experiment started (t = 0 h).
250 μl samples were taken at t = 0 h, t = 1 h, and t = 4 h; O.D. at 600 nm was measured:

	E37	E38	E39	E40	ENTERO4C5
t = 0 h	0.12	0.11	0.13	0.10	0.11
t = 1 h	0.22	0.15	0.21	0.28	0.19
t = 4 h	0.47	0.60	0.53	0.37	0.53

Then samples were centrifuged at 13.000 rpm; finally the supernatant was collected and stored at -20°C.
Glycerol stock for T9002-ENTERO4C5 was prepared.
Two 5 μl inocula of E32, E33, E34, E35, J101-E5, J101-31, J101-E6, J101-E7 and ENTERO4C5 were prepared in 1 ml of M9 + Cm12.5 for a preliminary test on TetR repressible promoter.

August, 4th

E41N plate was grown; three colonies were picked (E41N-1, E41N-2, E41N-3) and inoculated in LB + Cm12.5.
E32-1, E33-1, E34-1, E35-1, J101-E5-1, J101-31-1, J101-E6-1, J101-E7-1, E32-2, E33-2, E34-2, E35-2, J101-E5-2, J101-31-2, J101-E6-2, J101-E7-2, ENTERO4C5-1 and ENTERO4C5-2 were diluted 1:500 in a final volume of 1 ml. After 2 hours and 30 minutes inducible cultures were supplemented with anhydrotetracyclin (atc) at final concentrations of: 1 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml. Moreover uninduced cultures were supplemented with ddH₂O.
ENTERO-RBS and T9002-ENTERO4C5 were inoculated in 1 ml of Cm12.5 for preliminary AiiA test (with supernatants collected on August, 3rd).

August, 5th

ENTERO-RBS and T9002-ENTERO4C5 cultures were diluted 1:500 in a final volume of 1 ml and 10 ml of M9 + Cm12.5 respectively; they were grown for six hours. Plasmid purification was performed for E41N-1, E41N-2 and E41N-3:

Plasmid	DNA (ng/μl)
E41N-1	62.3
E41N-2	17.3
E41N-3	43.9

Screening digestion with EcoRI and PstI restriction enzymes was carried out:

Plasmid	DNA (μl)	H2O (μl)	Enzyme 1 (μl)	Enzyme 2 (μl)	Buffer H (μl)	Final Volume (μl)
E41N-1	2.5	19	0.5 EcoRI	0.5 Pstl	2.5	25
E41N-2	8.5	13	0.5 EcoRI	0.5 Pstl	2.5	25
E41N-2	3.5	18	0.5 EcoRI	0.5 Pstl	2.5	25

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.
A small-size agarose gel was prepared; in the afternoon gel electrophoresis was carried out:

Only E41N-1 showed the correct band, so we decided to keep it. E41N-2 and E41N-3 glycerol stocks were thrown.
TECAN test on E37, E38, E39, E40 and ENTERO4C5 supernatants was carried out; as supernatants were not diluted enough it was not possible to precisely measure 3OC₆-HSL concentration as the biosensor (T9002-ENTERO4C5) worked in the saturation zone.