Team:UNIPV-Pavia/Calendar/August/week1

August, 1st

Two colonies for E28-AGAIN and E41-AGAIN plates were picked and inoculated in LB + Cm1 12.5; J101-31, J101-E5 and J101-E7 plasmids were extracted:

Plasmid	DNA (ng/μl)
J101-31	???
J101-E5	???
J101-E7	???

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 Pstl	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; after 4 hours 2.5 μl of 2mM 3OC₆-HSL (1:2000 dilution) were added (final 3OC₆-HSL concentration 1μM) and the experiments starts (t = 0).

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