Team:UNIPV-Pavia/Calendar/August/week1

From 2011.igem.org

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<a name="August.2C_3rd"></a><h2> <span class="mw-headline">August, 3rd</span></h2>
<a name="August.2C_3rd"></a><h2> <span class="mw-headline">August, 3rd</span></h2>
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T9002-ENTERO4C5 plate was grown and a colony was picked and inoculated in LB + Amp + Cm12.5; E41N ligation was transformed in 100 &mu;l of MGZ1 competent cells and plated on a LB + Cm12.5 plate.
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34 LB agar + Cm12.5 plates were prepared according to protocols.
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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/&mu;l atc stock was prepared.
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After 3 hours cultures were induced with 5 &mu;l of atc; after 4 hours 2.5 &mu;l of 2mM 3OC<sub><small>6</small></sub>-HSL (1:2000 dilution) were added (final 3OC<sub><small>6</small></sub>-HSL concentration 1&mu;M) and the experiments starts (t = 0).
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<table border="0" width="100%" class="menu">
<table border="0" width="100%" class="menu">

Revision as of 20:51, 16 August 2011

UNIPV TEAM 2011

March
M T W T F S S
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31    

April
M T W T F S S
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30

May
M T W T F S S
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 31

June
M T W T F S S
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30

July
M T W T F S S
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31

August
M T W T F S S
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31

September
M T W T F S S
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30

October
M T W T F S S
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31

AUGUST: WEEK 1

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:

Medium size gel

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:

Smallsize gel

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).