Team:TU-Delft/results

Making knockout strain (far1::kanmx, gpa1::URA) and preparation of knocking out atf1

Week of 05-07-12

For making a functional knockout, yeast strains BY4741; Mat a; his3D1; leu2D0; met15D0; ura3D0; YJL157c::kanMX4 and BY4741; Mat a; his3D1; leu2D0; met15D0; ura3D0; YHR005c::kanMX4 were used (Euroscarf). Also knockout cassette pUG72 is used (Euroscarf). The LoxP-Ura-LoxP is elongated using the pFx polymerase protocol. The PCR program and primer sequences in table 1 yielded a product which is put on a gel shown in figure 1. Here a band can be recognized between 2000 and 1500 nucleotides, which corresponds to the 1669 nucleotide PCR product.

Table 1 PCR program for elongation of knockout cassette for knocking out Far1 and Gpa1 and primers used for elongation.

Repeats	Temperature		Duration
5x	95	Melting	2:00
	51	Annealing	1:00
	68	Elongation	2:00
25x	95	Melting	2:00
	61	Annealing	1:00
	68	Elongation	2:00

GPA ko fw TTAGCATCACATCAATAATCCAGAGGTGTATAAATTGATATATTAAGGTAGGAAATAATGCAGCTGAAGCTTCGTACGC

GPA ko rv TGCATCTTCGGAAACAGAATTTACGTATCTAAACACTACTTTAATTATACAGTTCCTTCAGCATAGGCCACTAGTGGATCTG

FAR1 ko fw ACACAAAGTCTATAGATCCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTAATGCAGCTGAAGCTTCGTACGC

FAR1 ko rv GAAAAAAAAAAAAGGAAAAGCAAAAGCCTCGAAATACGGGCCTCGATTCCCGAACTACTAGCATAGGCCACTAGTGGATCTG

GPA ko fw short TAATCCAGAGGTGTATAAATTGATATATTAAGGTAGGAAATAATGCAGCTGAAGCTTCGTACGC

GPA ko rv short AGAATTTACGTATCTAAACACTACTTTAATTATACAGTTCCTTCAGCATAGGCCACTAGTGGATCTG

FAR1 ko fw short ATCCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTAATGCAGCTGAAGCTTCGTACGC

FAR1 ko rv short AAAAGCAAAAGCCTCGAAATACGGGCCTCGATTCCCGAACTACTAGCATAGGCCACTAGTGGATCTG

ATF1 ko fw gaaaataaaaaacggCACTTCATCAGTATCACAAATACCATCAATTTATCAGCTCTCATGCAGCTGAAGCTTCGTACGC

ATF1 ko rv ggttatttacacgacatAATCATATTGTCGAATAATATCAGTCAAGCATCATGTGAGATCTAGCATAGGCCACTAGTGGATCTG

ATF1 ko fw short CACTTCATCAGTATCACAAATACCATCAATTTATCAGCTCTCATGCAGCTGAAGCTTCGTACGC

ATF1 ko rv short AATCATATTGTCGAATAATATCAGTCAAGCATCATGTGAGATCTAGCATAGGCCACTAGTGGATCTG

Figure 1 1% agarose in TAE ~45 run on 80 Volts. In the picture can be seen: 1 SmartLadder, 2 Far1 short PCR product, 3 Far1 long PCR product, 4 Gpa1 short PCR product, 5 Gpa1 long PCR product, 6 Atf1 short PCR product, 7 Atf1 long PCR product.

Gel extraction of the GPA1 PCR product is performed using the Qiagen gel extraction kit. The end concentration of this step is measured to be 167.2 ng/µl using the nanodrop nucleotide program.

The PCR reactions using FAR1 and ATF1 primers with the same conditions as table 1 but with the PCR program according to table 2 is performed yielding no result.

Table 2 PCR program for elongation of knockout cassette for knocking out Far1 and Gpa1 and primers used for elongation.

Repeats	Temperature		Duration
30 x	95	Melting	2:00
	60/62/66/69	Annealing	1:00
	68	Elongation	2:00

Drop out medium is made and solutions for transformations in yeast strains is prepared. The formula of the dropout media can be seen in the media protocol.

Week of 09-07-12

Yeast strain BY4741; Mat a; his3D1; leu2D0; met15D0; ura3D0; YJL157c::kanMX4 is transformed with the GPA1 ko PCR product, according to the yeast transformation protocol. 100 ng of DNA is used for transformation. After 3 days 3 colonies could be seen on the plate. There where zero colonies on the control plate.
To check whether the knock-out reaction was successful, colony PCR of the three colonies where performed. Primers A and D bind to 5’ upstream and 3’ downstream regions of the GPA1 gene, the B and C primers anneal in the URA gene which is knocked in. For the first reaction, only AD reaction is tried A negative control is the yeast strain without transformation. Before initiation the colonies where boiled for 10 minutes in a PCR tube in 10 µl 2 mM NaOH solution of which 2 µl is taken. First conditions according to table 3 where used, using taq Mastermix PCR solution. This yielded no results when put on gel.

Tabel 3 PCR conditions of knock-out PCR check

Repeats	Temperature		Duration
	94		Melting	2:00
30x	94		Melting	1:00
	58		Annealing	2:00
	72		Elongation	2:00
	72		Elongation	10:00

GPA1 A (fw)TCTGCGTATTCTTCCTTGTAGAAAT

GPA1 D (rv) GAATTCGAGATAATACCCTGTCCTT

A second run with conditions according to table 4 was performed. Now colonies where dissolved in 20 µl 2 mM NaOH of which 18 µl was taken (the maximum amount). This yielded no results when put on gel.

Table 4 PCR on pUG73 cassette with Phusion polymerase summary.

Repeats	Temperature		Duration
	94	Melting	2:00
25x	94	Melting	1:00
	72	Elongation	4:00
		72	Elongation	10:00

GPA1 A (fw) TCTGCGTATTCTTCCTTGTAGAAAT

GPA1 D (rv) GAATTCGAGATAATACCCTGTCCTT

Week of 18-07-12

A new PCR kit, phusion polymerase, is tried. PCR conditions and mix are shown in table 5.

Table 5 colony PCR on WT yeast with Phusion polymerase summary.

Repeats	Temperature		Duration
	98	Melting	0:30
25x	98	Melting	0:20
	66	Annealing	0:30
	72	Elongation	2:30
	72	Elongation	10:00

GPA1 A (fw) TCTGCGTATTCTTCCTTGTAGAAAT
GPA1 D (rv) GAATTCGAGATAATACCCTGTCCTT
Phusion mix
5x Phusion buffer	10 µl
10 mM dNTP’s	1 µl
Primer A	5 µl
Primer D	5 µl
DNA template (boiled)	2 µl
DMSO	1.5 µl
Polymerase	0.5 µl
H2O	25 µl