ACCIONES JURÍDICAS
2. Principales repertorios de acción colectiva utilizados por La Mesa y CDD
was purified (EZNA Cycle-‐Pure kit, Omega Bio-‐tek) and mixed with 0.2 µl 10x PCR
Thermo buffer, 0.2 µl Taq polymerase (5.0 U µl-‐1), 0.4 µl 10 mM dATPs for a final
concentration of 0.2 mM, to a final volume of 20 µl. The reaction was incubated for
15 minutes at 72°C and the product subsequently ligated with pCR8 following the
standard protocol as follows (Invitrogen): 4 µl of the PCR product was incubated
with 1 µl salt solution and 1 µl TOPO vector for 5 minutes at room temperature
and then placed on ice. Two µl of the TOPO ligation mixture was added to a vial of thawed One-‐shotTM chemically competent cells and incubated on ice for 25
minutes. After exposing the cells to a heat shock for 30 sec at 42°C, 250 µl of SOC
medium was added. After one hour incubation at 37°C, 25 µl, 50 µl and 75 µl
aliquots of bacterial culture were spread on LB-‐agar plates containing spectinomycin, and incubated overnight at 37°C. Colonies were selected from the
plate the following day and sent for sequencing to confirm that the carB* insert was introduced into the plasmid without any additional mutations. The strains were stored at -‐80°C.
2.4.3 Cloning into the pUIC3 plasmid
E.coli carrying pCR8-‐carB* was grown in 5 ml LB overnight in the presence of spectinomycin. After purification (EZNA Plasmid mini kit, Omega Bio-‐tek) the plasmid was digested with Bgl II. In a total volume of 50 µl, 1 µg of vector DNA was
mixed with 5 µl 10x NEB 3 buffer, 1 µl Bgl II enzyme (10 U µl-‐1) and H2O and
incubated for 1-‐2 hours at 37°C. The fragment was separated from the vector by
gel electrophoresis in a 1% TAE agarose gel at a voltage of 100, cut out of the gel and purified. The product was stored at -‐20°C until ligation with plasmid pUIC3.
The suicide reporter plasmid pUIC3 contains a tetracycline resistance gene, a promoter less lacZY operon and a multiple cloning site (Rainey, 1999). The vector is relatively large and as a consequence a higher volume of cell culture must be mixed with the plasmid to obtain a sufficient yield of transformants. Therefore,
two 5 ml LB overnight cultures were inoculated with E.colipUIC3 and incubated at 37°C overnight. After plasmid purification 2 µg of plasmid DNA was linearised by
digestion with 2 µl Bgl II (10 U µl-‐1), 10 µl 10x NEB 3 buffer, and H20 to a final
volume of 100 µl. The linearised vector was isolated by gel electrophoresis in a 1%
TAE gel and purified. To prevent self-‐ligation the digested vector was treated with Antarctic phosphatase to remove 5’-‐phosphate groups from the ends: 1 µg of
linearised vector DNA was mixed with 0.5 units of CIP and incubated at 37°C for
one hour. The product was cleaned with a PCR clean up kit (EZNA Cycle-‐Pure kit, Omega Bio-‐tek) and stored at -‐20°C.
Before ligating the carB fragment with pUIC3 the concentration of both insert and vector were estimated by spectrometry (BioPhotometer, Eppendorf). The ligase reaction contained a molar ratio of 3:1 of insert to vector: 1.45 µl of linearised
pUIC3 was added to 7.05 µl of the carB fragment, 1.0 µl 10x NEB buffer, and 0.5 µl
ligase, and incubated at 16°C overnight. DNA ligase was inactivated by heating the
reaction to 65°C for 20 minutes, and stored at 4°C until transformation into E. coli.
2.4.3.1 Manufacture of electrocompetent cells
Electrocompetent E.coliDH5αλpir were prepared by the method of Sambrook and Russell (Sambrook & Russell, 2001). The starter culture, 5 ml LB, was inoculated from the freezer stock and grown overnight at 37°C. 3 ml of the starter culture was
used to inoculate a flask with 350 ml LB that was pre-‐heated to 37°C. The OD600 of
the culture was monitored every 15-‐20 minutes. When OD600 reached between
~0.35-‐0.4, the flask was transferred to ice for 20 minutes to stop further growth. Then, the culture was decanted into six x 50 ml chilled centrifuge tubes. The cells were pelleted at 4°C, at 900 x g for 15 minutes. The supernatant was discarded and
the cells were washed with 50 ml chilled H2O. The cells were pelleted at 4°C, at 900
x g for 15 minutes and the supernatant was discarded. The second wash followed the same procedure. In the final wash the pellets were resuspended in 50 ml of chilled 10% glycerol, pooled in one tube that was centrifuged at 4°C, at 900 x g for
15 minutes, and the supernatant was discarded. The pellet was resuspended in the residual liquid and aliquots of 50 µL were transferred into pre-‐chilled
microcentrifuge tubes and stored at -‐80°C.
2.4.3.2 Transformation of pUIC3 into E. coli
An aliquot of E.coliDH5αλpir electrocompetent cells was thawed on ice and subsequently mixed with 2.5 µl of the pUIC3carB*-‐construct. The mixture was
pipetted into a pre-‐chilled 2 mm cuvette that was inserted into an Elecroporator (Electroporator 2510; Eppendorf). The voltage was set at 2.5 KV and after electroporation 500 µl SOC were immediately added to the cell suspension. The
culture recovered for one hour at 37°C and was then plated on LB agar plates
containing Ampicillin and X-‐gal. The next day several blue colonies were selected from the plate and a PCR was performed using vector specific primers. Colonies that showed a correctly-‐sized insert were inoculated into LB broth, grown overnight at 37°C, and stored at -‐80°C.
2.4.4 Bacterial conjugation
The carB* mutation was introduced into P. fluorescens through tri-‐parental mating. Conjugative plasmid transmission from the donor strain E.colipUIC3carB* to a P. fluorescens recipient strain was assisted by the Km resistant helper plasmid
pRK2013, which provided mob and tra functions in trans (Ditta et al., 1980). The donor and the helper were grown overnight in 5 ml LB with the appropriate antibiotics, and the recipient without any antibiotics. The next day the OD600 was
measured to ensure equal amounts of each culture to optimise pUIC3carB* transmission frequency. Approximately 700 µl of the recipient was subjected to
heat shock conditions at 45°C for 20 minutes. 300 µl of the donor and helper