Team:Slovenia/METHODS and PARTS/notebook/oscil

8/16/2010-8/22/2010

Primers for T7 RNA polymerase, TAL, mCherry, Zif268, Tet R and hybrid promoters (pT7_TetO, pT7_ZIF268) were ordered. pSB1AK8 backbone vector containing CMV promoter was cut with EcoRI/SpeI and SpeI/PstI restriction enzymes. The fragments were isolated from agarose gel.

8/23/2010-8/29/2010

Primers for the Smolen oscilator have arrived. Annealing of hybrid promoter primers was performed. Also, PCR was performed on T7 RNA polymerase, TAL, Tet represor, mCherry, ZIF268 zinc finger, PEST and CL1-PEST sequence.  Hybrid promoters were ligated into the pSB1AK8 vector. Control restriction was done for verification.

8/30/2010-9/5/2010

Plasmids containing hybrid promoters were cut with SpeI/PstI restriction enzymes. T7 RNA polymerase, TAL, Tet represor, mCherry and ZIF268 were ligated behind the hybrid promoter. Ligation of T7 RNA polymerase was unsuccessful and had to be repeated.

9/6/2010-9/12/2010

PEST and CL1-PEST fast degradation sequence were ligated behind T7 RNA polymerase, TAL, Tet represor, mCherry and ZIF268 zinc finger.

9/13/2010-9/19/2010

Primers for pcDNA3 vector modification have arrived. PCR was performed with Phusion polymerase. SsrA degradation tags were ligated behind Tet represor and TAL. Ter represor, TAL and T7 RNA polymerase were ligated behind CMV promoter. PCR was performed on Venus reporter protein. Tet represor, TAL and T7 RNA polymerase were ligated in front of T7 terminator. Modified pcDNA3 vector was cut with XbaI and XhoI restriction enzymes.

9/20/2010-9/26/2010

Venus reporter protein was cloned into pSB1AK8 vector. pT7_TetO hybrid promoter was ligated in front of Venus. Venus with SsrA degradation tag was ligated in front of T7 terminator. All constructs in pSB1AK8 vector were PCRed out of pSB1AK8 vector together with pT7_TetO hybrid promoter and mammalian terminator.

9/27/2010-10/3/2010

PCR products were cut with XbaI and SalI restriction enzymes and ligated into the modified pcDNA3 vector. Annealing of primers for pT7_TetO hybrid promoter was repeated and ligated in front of Venus.

4/10/2010-10/10/2010

A series of tests were performed in HEK293 cells and shot under a confocal microscope. HEK293 cells were transfected with Venus under pT7_TetO promoter and T7 RNA polymerase under CMV promoter. Also HEK293 cells were transfected with Venus under pT7_TetO, T7 RNA polymerase and  TAL or Tet repressor under CMV promoter.  Venus under pT7_TetO promoter and T7 RNA polymerase under CMV promoter were compared with mCerrulean under pT7 promoter and T7 RNA polymerase under CMV promoter.

10/11/2010-10/17/2010

Venus under pT7_TetO hybrid promoter was transformed into BL21 cells. Different amounts of Venus under pT7_TetO promoter and T7 RNA polymerase under CMV promoter were transfected into HEK239 cells and shot under confocal microscope. Also HEK239 were transfected with Venus under pT7_Teto promoter and different amounts of T7 RNA polymerase under CMV. Venus was cloned into pGL4.16 vector and tested. Genes for represilator have arrived. New primers for Venus have arrived.

10/18/2010-10/24/2010

Represilator genes were transformed into DH5α and BL21 cells. PCR was performed on Venus with new primers. PCR product was cut with BsiWI and KasI restriction enzymes and cloned into the first represilator gene. Constructs in the pSB1AK8 vector were cut with EcoRI and PstI restriction enzymes and ligated into pSB1C3 vector. HEK293 were transfected with mCherry under pT7 promoter, T7 RNA polymerase under pT7_TetO and CMV promoter and TAL under pT7_TetO promoter.