Ricinus cDNA library screening

B.2.21.1. Yeast Transformation

5 ml of YPD liquid medium was inoculated with the invertase deficient yeast strain SEY2102and incubated overnight on a rotary shaker at 200 rpm at 30°C. After that, 2.5 x 10⁸ cells were added to pre-warmed YPD to give 5 x 10⁶ cells/ml. Then the flask was incubated on a rotary or reciprocating shaker at 30°C and 200 rpm. When the cell titer was at least 2 x 10⁷ cells/ml, the cells were harvested by centrifugation at 3000 g for 5 min.

Afterwards the cells were washed in 25 ml of sterile water and resuspended in 1 ml of sterile water. The cell suspension was transferred to a 1.5 ml centrifuge tube and centrifuged for 30 sec. Sterile water was added to a final volume of 1.0 ml to the cell pellet and then the cells were resuspended in sterile water. After that, 100 µl of cell suspension was added (ca. 10⁸ cells) into 1.5 ml microfuge tubes. Then the tube was centrifuged at top speed for 30 sec and the supernatant was removed. Then the reagents were added as follow:

Reagents:

The transformation mix was mixed well and incubated in a 42°C water bath for 40 min.

After that, the transformation mix was centrifuged at top speed for 30 sec. The supernatant was removed with a micropipettor. Subsequently, 1.0 ml of sterile water was added to tube to resuspend the cell pellet. Then 200 µl of cell suspension was plated on to the MMA/Leucine/Histidine plate. The plate was incubated at 30°C for 4 days. The transforment was named SEYSC1.

B.2.21.2. cDNA library screening

SEYSCR1 could produce RcSCR1 protein and transport sucrose into cell when it grew in a sucrose-containing medium, SEYNEV could not produce RcSCR1 protein and no sucrose could be transported into cell when it grew in a sucrose-containing medium. Then SEYSCR1 and SEYNEV were transformed with 181A1NE plasmid. The new double transformants, which had nSC4+/181A1NE or NEV-N/181A1NE, were used as negative control and positive control. The positive control (SEYNEV::181A1NE) could grow better than negative control (SEYSCR1::181A1NE). The negative control grew better in a MMA medium without sucrose (Figure 12). If sucrose efflux transporter can release sucrose to the medium and sucrose might be not accumulated too high in yeast. These transformants might grow better than other transformants, which could not transport sucrose to medium.

That is the basic theory of screening.

In order to find sucrose efflux transporters from Ricinus endosperm, an expression cDNA library was used. The expression cDNA library was constructed by Dolle (Dolle, diploma thesis, Bayreuth 1994). The Ricinus seedling cDNA was ligated to a yeast expression vector 181A1NE, which contains Adh1 promoter and Adh1 terminator. The screening was performed as following (Figure 13): the plasmid, which contains cDNA, was transformed to SEYSC1. After transformation, the yeast cells were plated on an MMA/Histidine plate and

incubated at 30^oC for 4 days. After that, 5 ml of MMA were used to wash out the yeast transforments. 4 ml of the transforments was transferred to a flask, which contains 100 ml of 2% sucrose in MMA/Histidine. The rest of the transforments were transferred to a centrifuge tube and an equal volume of sterile 1M Sorbitol was added. The tube was kept in -80^oC.

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 Time (hour)

OD 600

nSC4+::181A1NE, no sucrose nSC4+::181A1NE, 2% sucrose NEV-N::181A1NE, 2% sucrose

Figure 12 : The growth curves of the negative control and positive control. The positive control was cultured in a 2% sucrose -containing MMA medium. The negative control was cultured in a 2%

sucrose -containing MMA medium or in a MMA medium. The positive control can grow quicker than the negative control.

The transforments in sucrose/MMA/Histidine selection medium were then incubated in a shaking incubator at 200 rpm, 30^oC for 8 hours. After that, the culture was centrifuged and resuspended in 100 ml of sucrose/MMA/Histidine medium. The selection medium was changed every 8 hours for 7 days. After that, the yeast cells were collected and plated to the MMA/Histidine plate.

When the colonies could be seen on the plates, the single colonies were picked and put into 3 ml of MMA/Histidine medium. The yeast was then incubated in a shaking incubator at 200 rpm, 30^oC overnight. 1ml of these overnight culture s was used to measure the OD600. Then all the overnight cultures were diluted to the same concentration (OD600=0.1).

200 µl of each diluted culture was transferred to a microtiter plate and put into ELISA reader to measure the growth curves.

A B

Figure 13 : The basic theory of sucrose efflux screening. A. nSC4+ transformed yeast (SEYSCR1) grows slower in a 2% sucrose containing MMA. B. The flowchart of the screening.

The growth curves of the different transforments were compared to the growth curve of the negative control. The transformants, which could growth quicker then control in a MMA/

2% sucrose/ Histidine medium, were then cultured. The plasmids from these transforments were extracted and transformed to E.coli DH5α. The E.coli transforments were then

checked by PCR. The transforments, which did not contain nSC4+ sequence, were kept for later study.

B.2.21.3. Quick Preparation of Plasmid DNA from Yeast

2 ml of yeast liquid culture was collected and resuspend in 500 µl of lysis buffer (50mM Tris-HCl, pH 7.5, 10mM EDTA, 0.1% SDS and 10mM 2-mercaptoethanol added just before use). Sample was mixed vigorously by vortexing. An equal volume of glass beads (0.45 mm) was added and vortexed at top speed for 1 min. Then an equal volume of phenol/CHCl3 was added. The aqueous phase was transferred to a new tube and extracted with CHCl3 one time. DAN was precipitated by adding 50 µl of 3M NaOAc and 500 µl of isopropanol. Subsequently, DNA was washed once with 80% ETOH and air-dried.

Then DNA was resuspended in 100 µl of TE and use to transform E. coli DH5α.