Team:Slovenia/METHODS and PARTS/notebook/znf

5/31/2010 – 6/5/2010

DH5α cells were transformed with pSB1AK3 plasmid containing RBS-lacZ(full) insert and plated on LB with ampicylin (Amp). Single colonies were later inoculated into liquid LB media with Amp. Plasmid DNA was isolated from the over-night culture. The plasmid was cut with EcoRI and SpeI enzymes and the RBS-lacZ(full) fragment was isolated from agarose gel and ligated into pSB1AK3 vector containing DTER, which was previously cut with EcoRI and XbaI enzymes and isolated from the gel.

6/6/2010 – 6/12/2010

A synthetic promotor pSYN_BbsI was ligated into pSB1AK3 vector with DTER. DH5α cells were transformed with the ligation product and put on LB Amp agar plates. Single colonies were later inoculated into liquid LB media with Amp and plasmid DNA was isolated from the over-night cultures. Restriction analysis with EcoRI and PstI enzymes was performed.

pSB1AK3 plasmid containing the RBS-lacZ(full)-DTER insert was cut with EcoRI and XbaI enzymes and the vector was then isolated from the gel.

6/13/2010 – 6/19/2010

pSB1AK3 plasmid with DTER-pSYN_BbsI was cut with BbsI enzyme and isolated from the gel. Primer annealing was done for the Gli1, HivC and Tet operators, and the fragments were ligated into the cut vector. DH5α cells were transformed with the ligation product and plated on LB Amp.

6/14/2010 – 6/20/2010

Colony PCR was performed to ascertain which colonies contained plasmids coding for Zinc-fingers and Tet operators. Positive colonies were later inoculated into liquid LB media with Amp and plasmid DNA was isolated from the over-night cultures and sent to sequencing.

6/21/2010 - 6/27/2010

Sequencing results showed that the ligations of Zinc-finger operators were succesful, unlike the ligation of Tet operator. Primer annealing was performed again and the whole process was repeated. DB3.1 cells were transformed with pSB4C5 plasmid, which was taken from the Registry. The cells were grown on LB chloramphenicol (Cm) agar plates and later inoculated into liquid LB media with Cm.

6/28/2010 – 7/4/2010

pSB1AK3 vector containg BsaI_DTER was cut with EcoRI and XbaI enzymes and isolated from agarose gel. pBAD/araC fragment, which was previously cut with EcoRI and SpeI enzymes and isolated from the gel, was ligated into the latter vector and DH5α cells were transformed with the ligation products and plated on LB Amp. Single colonies were later inoculated into liquid LB media with Amp and plasmid DNA was isolated from the over-night cultures. Restriction analysis for the construct pBAD/araC-BsaI-DTER was performed with the enzymes EcoRI and PstI.

pSB4C5 plasmid was isolated from the over-night culture and restriction analysis with the enzyme AvaI was performed.

Sequencing results showed that the second ligation of Tet operator into the pSB1AK3 vector containing DTER-pSYN_BbsI was unsuccesful again. Another set of primers for annealing of the Tet operator were designed and ordered. Alongside these, primers for annealing of ATG and the rest of the Zinc-finger operators were designed and ordered.

7/5/2010 – 7/11/2010

The pBAD/araC-BsaI-DTER fragment was cut with XbaI and PstI enzymes and isolated from the gel. The fragment was then ligated into the pSB1AK3 vector containing RBS-lacZ(full)-DTER, which was previously cut with SpeI and PstI enzymes and isolated from the gel. DH5α cells were transformed with the ligation products and plated on LB Amp. Single colonies were later inoculated into liquid LB media with Amp. Plasmid DNA was isolated from the over-night cultures and restriction analysis with EcoRI and PstI enzymes was performed. Restriction of pSB4C5 plasmid with EcoRI and PstI was performed. The plasmid backbone was isolated from the gel and stored for later use.

7/12/2010 - 7/18/2010

Primers with ATG arrived. Annealing of ATG primers was done and the fragment was then ligated into pSB1AK3 vector. DH5α cells were transformed with the ligation product and plated on LB Amp. Colony PCR was performed and positive colonies were inoculated into liquid LB media with Amp and plasmid DNA was later isolated and sent to sequencing. pSB1AK3 vectors with STOP and His STOP were cut with EcoRI and XbaI enzymes and pSB1AK3 vector with ATG His was cut with SpeI and PstI enzymes. All plasmid backbones were later isolated from the gel.

7/19/2010 – 7/25/2010

Primers with Zinc-finger and Tet operators finally arrived! Annealing of the primers was done and the fragments were then ligated into pSB1AK3 vector with DTER-pSYN, which was previously cut with BbsI enzyme and isolated from the gel. DH5α cells were transformed with the ligation products and plated on LB Amp. Colony PCR was performed and positive colonies were inoculated into liquid LB media with Amp and plasmid DNA was later isolated and sent to sequencing.

At the end of the week, a package from GeneArt with our Zinc-fingers came, which were transformed into DH5α cells. Colonies grown on LB Amp agar plates were later inoculated intoliquid LB media with Amp and plasmid DNA was isolated from the over-night cultures.

7/26./2010 – 8/1/2010

Plasmids with Zinc-fingers were cut with EcoRI/SpeI and XbaI/PstI enzymes. The Zinc-finger fragments were then isolated from the gel and ligated into pSB1AK3 vectors with ATG His, STOP and His STOP. DH5α cells were transformed with the ligation product and plated on LB Amp. Colony PCR was performed and positive colonies were later inoculated into liquid LB media with Amp and plasmid DNA was isolated from the over-night cultures.

Sequencing results showed that all ligations of Zinc-finger operators were unsuccessful. The pSB1AK3 vector with DTER-pSYN was cut with BbsI enzyme and isolated from the gel again and primer annealing was repeated. The fragments were then ligated and transformed into DH5α cells, which were grown on LB Amp agar plates. Colony PCR was performed and positive colonies were inoculated into liquid LB media with Amp and plasmid DNA was isolated from the over-night cultures and sent to sequencing again.

However, the ligation of ATG into pSB1AK3 turned out to be successful.

8/2/2010 – 8/8/2010

Plasmids containing Zinc-fingers with STOP and His STOP were isolated from over-night cultures. They were later cut with XbaI and PstI enzymes and the Zinc-finger fragments were isolated from gel.

pSB1AK3 vector with ATG was cut with SpeI and PstI enzymes and isolated from the gel. Zinc-finger fragments were ligated into the vector and transformed into DH5α cells, which were grown on LB Amp agar plates. Colony PCR was performed and positive colonies were later inoculated intoliquid LB media with Amp and plasmid DNA was isolated from the over-night cultures.

Sequencing results showed that 4 out of 5 ligations of Zinc-finger operators and even Tet operator were successful this time. Another primer annealing was done for the Tyr456 operator, and the whole process was repeated. The plasmid supposedly containing the Tyr456 operator was sent to sequencing.

8/9/2010 – 8/15/2010

Zinc-finger fragments with STOP were ligated into pSB1AK3 vectors with ATG and ATG His, and Zinc-finger fragments with His STOP into pSB1AK3 vector with ATG. DH5α cells were transformed with the ligation products and plated on LB Amp. Colony PCR was performed and positive colonies were later inoculated into liquid LB media with Amp. Plasmid DNA was isolated from the over-night cultures. pSB1AK3 plasmids containing DTER-pSYN with Zinc-finger and Tet operators were cut with EcoRI and SpeI enzymes. The fragments were then isolated from the gel and stored for later use.

8/16/2010 – 8/22/2010

The RBS-LacZfull-DTER-pBAD-BsaI-DTER construct was cloned into the pSB1C3 vector and was later cut with the BsaI enzyme and the fragment was isolated from the gel. The TetR fragment, which was cut with XbaI and PstI enzymes and isolated from the gel, was ligated into the previously cut pSB1AK3 vector containing ATG. DH5α cells were transformed with the ligation products and plated on LB Amp. Colonies were inoculated into mini-prep flasks with Amp and plasmid DNA was isolated from the over-night cultures. Restriction analysis was performed with EcoRI and PstI enzymes. The plasmid was then cut with EcoRI and SpeI enzymes and the ATG-TetR fragment was isolated from the gel and later ligated into the previosly cut pSB1AK3 vector containing STOP. DH5α cells were transformed with the ligation products and plated on LB Amp. Colonies were inoculated into liquid LB media with Amp and plasmid DNA was isolated from the over-night cultures. Restriction analysis was performed with EcoRI and PstI enzymes.

8/23/2010 – 8/29/201

ATG-TetR-STOP and Zinc-finger constructs containing ATG His and STOP were cut with XbaI and NotI enzymes and isolated from the gel. The fragments were then ligated into the pSB1C3 vector containing RBS-LacZfull-DTER-pBAD-BsaI-DTER. DH5α cells were transformed with the ligation products and plated on LB Cm. Colonies were inoculated into liquid LB media with Cm and plasmid DNA was isolated from the over-night cultures. Restriction analysis was performed on the isolated plasmids.

8/30/2010 – 9/5/2010

Restriction analysis was performed on the isolated plasmids supposedly containing RBS-LacZfull-DTER-pBAD-Zinc-finger/TetR-DTER. Some ligations were unsuccessful, so the whole process for those constructs was repeated.

9/6/2010 – 9/12/2010

Same as above.

9/13/2010 -9/19/2010

The first test for in vivo binding of Zinc Fingers to synthetic promoter was performed. We used beta-galactosidase as our reporter (for more detailed protocols read page »Methods«).

9/20/2010 - 9/26/2010

Optimization of method for testing in vivo binding of Zinc Fingers.