Función de conocimiento : Permite comprender y explicar la realidad Las representaciones permiten a los actores sociales adquirir nuevos conocimientos e

pOFX-lac1TF and pOFX-lac1TFNC

The ORFs for the TF and TFNC were PCR amplified using 5’-primer encoding XbaI and NdeI (coding for initiator Methionine) sites and 3’-primer encoding BamHI site from pProEX-TF and pProEX-TFNC plasmids (Kaiser). The reactions were purified and digested with XbaI and BamHI. The digested inserts were purified and eluted in NF water. The empty plasmid pOFX-lac1 was digested with XbaI and BamHI and subsequently dephosphorylated with calf intestinal alkaline phosphatase (CIAP). The vector backbone was purified and eluted with NF water. For the ligation reaction, both

the purified plasmid and insert were incubated with 400 units of T4 DNA ligase at 16 °C for 16 h in a ratio of 1:3. In a control reaction NF water was added instead of the insert. The whole ligation reaction was transformed into chemically competent DH5α cells and plated on LBSpec plates and incubated overnight at 37 °C. Single colonies were inoculated in LBSpec for overnight cultures. Cultures were harvested and plasmids were prepared. The presence of the insert was confirmed by restriction digestion with XbaI and BamHI. Positive clones were confirmed additionally by sequencing the DNA.

pBAD18-Ras-DHFR-His6

The ORF of Ras-DHFR-His6 was excised from pET3a-Ras-DHFR- His6 using

XbaI and HindIII restriction enzymes and purified. pBAD18-Luc was digested with XbaI and HindIII to release Luc and the plasmid backbone was purified from Agarose gel. For the ligation reaction, both the purified plasmid backbone and insert were incubated with 400 units of T4 DNA ligase at 16 C for 16 h in a ratio of 1:3. In a control reaction NF water was added instead of the insert.

The whole ligation reaction was transformed into chemically competent DH5α cells and plated on LBAmp plates and incubated overnight at 37 °C. Single colonies were inoculated in LBAmp for overnight cultures. Cultures were harvested and plasmids were prepared. The presence of the insert was confirmed by restriction digestion with XbaI and HindIII. Positive clones were confirmed additionally by sequencing the DNA.

Plasmids for the co-expression system

The ORF of Luc was excised from pBAD18-Luc using XbaI and HindIII restriction enzymes and purified. pBAD33 was digested with XbaI and HindIII, followed by dephosphorylation with CIAP and purification. For the ligation reaction, both the purified plasmid backbone and insert were incubated with 400 units of T4 DNA ligase at 16 C for 16 h in a ratio of 1:3. In a control reaction NF water was added instead of the insert. The whole ligation reaction was transformed into chemically competent DH5α cells and plated on LBCm plates and incubated overnight at 37 °C. Single colonies were

inoculated in LBCm for overnight cultures. Cultures were harvested and plasmids were prepared. The presence of the insert was confirmed by restriction digestion with XbaI and HindIII. Positive clones were confirmed additionally by sequencing the DNA.

Human Hsc70 (hHsc70) was PCR amplified using 5’-primer encoding NcoI (coding for initiator Methionine) site and 3’-primer encoding NotI site from the pET11a- Hsc70 plasmid (Lab collection). The reaction was purified and digested with NcoI and NotI. The digested inserts were purified and eluted in NF water. The empty plasmid pCDFDUET (Novagen) was digested with NcoI and NotI and subsequently dephosphorylated and purified. For the ligation reaction, both the purified plasmid and insert were incubated with 400 units of T4 DNA ligase at 16 C for 16 h in a ratio of 1:3. In a control reaction NF water was added instead of the insert. The whole ligation reaction was transformed into chemically competent DH5α cells and plated on LBSpec plates and incubated overnight at 37 °C. Single colonies were inoculated in LBSpec for overnight cultures. Cultures were harvested and plasmids were prepared. The presence of the insert was confirmed by restriction digestion with NcoI and NotI. Positive clones were confirmed additionally by sequencing the DNA.

Human Hdj2 (hHdj2) was excised from the pCH-Hdj2 plasmid (Lab collection) using NdeI and NheI restriction enzymes and purified. pETDUET (Novagen) was digested with NdeI and BlnI, followed by dephosphorylation with CIAP and purification. For the ligation reaction, both the purified plasmid backbone and insert were incubated with 400 units of T4 DNA ligase at 16 C for 16 h in a ratio of 1:3. In a control reaction NF water was added instead of the insert. The whole ligation reaction was transformed into chemically competent DH5α cells and plated on LBAmp

plates and incubated overnight at 37 °C. Single colonies were inoculated in LBAmp for overnight cultures. Cultures were harvested and plasmids were prepared. The presence of the insert was confirmed by restriction digestion with BamHI. Positive clones were confirmed additionally by sequencing the DNA.

TFNC was PCR amplified using using 5’-primer encoding NdeI (coding for initiator Methionine) site and 3’-primer encoding BlnI site from the pPROEX-HTa-

TFNC plasmid (Lab collection). The reaction was purified and digested with NcoI and NotI. The digested inserts were purified and eluted in NF water. The empty plasmid pCOLADUET (Novagen) was digested with NdeI and BlnI and subsequently dephosphorylated and purified. For the ligation reaction, both the purified plasmid and insert were incubated with 400 units of T4 DNA ligase at 16 C for 16 h in a ratio of 1:3. In a control reaction NF water was added instead of the insert. The whole ligation reaction was transformed into chemically competent DH5α cells and plated on LBKan plates and incubated overnight at 37 °C. Single colonies were inoculated in LBKan for overnight cultures. Cultures were harvested and plasmids were prepared. The presence of the insert was confirmed by restriction digestion with NdeI and BlnI.

This plasmid was further used for cloning of human Bag1 (hBag1) in the MCSI by digesting with NcoI and Ecl136II. Bag1 was excised from the pROEX-HTa-Bag1 plasmid (Lab collection) using NcoI and MscI restriction enzymes and purified. For the ligation reaction, both the purified plasmid backbone and insert were incubated with 400 units of T4 DNA ligase at 16 C for 16 h in a ratio of 1:3. In a control reaction NF water was added instead of the insert. The whole ligation reaction was transformed into chemically competent DH5α cells and plated on LBKan

plates and incubated overnight at 37 °C. Single colonies were inoculated in LBKan for overnight cultures. Cultures were harvested and plasmids were prepared. The presence of the insert was confirmed by restriction digestion with NcoI and HindIII. Positive clones were confirmed additionally by sequencing the DNA.