Transformation of competent cell

The competent cell in the eppendorf tube from -80^oC was put on the ice for unfreezing and then added 0.1~1μg plasmid DNA. After ice bath for 30 minutes, the competent cell was heat shock at 42^oC for 1 minute and then added 300μl LB broth with 50μg/ml Ampicillin. The cell solution was incubated at 37^oC for 1 hours with vigorous shaking (~180 rpm/minutes). Each 100μl of the cell solution was dispread on the LB agar medium with 50μg/ml Ampicillin and incubated at 37^oC for 12~18 hours.

3.2 Isolation of E.coli plasmid DNA

The plasmid in E.coli was extracted with ExcelPure^TM Plasmid Mini kit (Premier, Cat. No.PM250). The procedure is as following :

The DH5α with target plasmid was inoculated into 5 ml LB broth with 50μg/ml Ampicillin and incubated at 37^oC for 16~18 hours with vigorous shaking (~180 rpm/minutes). 1.5 ml of cell solution was transferred to an eppendorf tube. The cells were recovered by centrifugation at 13000 rpm for 1 minute and then resupended in 200μl Solution I buffer (Premier, Cat. No.PM250). Then 200μl of Solution II buffer (Premier, Cat. No.PM250) was added to the mixture and mixed gently. Initially, 200μl of Solution III buffer (Premier, Cat. No.PM250) was added to the mixture and mixed gently again. The mixture was centrifuged at 13000 rpm for 5 minutes at room temperature. The supernatant was transferred to Mini-M^TM Column (Premier, Cat.

No.PM250) packed with collection tube and spun at 13000 rpm for 1 minute. The filtrate in the collection tube was discarded. 700 μl of Wash Solution (Premier, Cat.

No.PM250) was added to Mini-M^TM Column and spun at 13000 rpm for 1 minute.

The filtrate was discarded and the washing step was repeated once again. After the filtrate was discarded, the column was centrifuged at 13000 rpm for 3 minutes to remove residual ethanol. The Mini-M^TM Column was transferred to a new eppendorf tube and incubated at 60^oC for 5 minutes to evaporate the ethanol. Finally, the DNA in the column was eluted in 50μl ddH2O by spinning at 13000 rpm for 1 minute. The plasmid DNA was stored at -20^oC.

3.3 Restriction enzyme digestion

The plasmid DNA was digested into DNA fragment by restriction enzyme. The

reaction mixture was added by 0.5~10μg plasmid DNA, 0.1~1μl restriction enzyme and reaction buffer to appropriate volume (10~50μl). The reaction for each kind of restriction enzyme was according to the recommend condition in operation manual.

After the reaction was finished, the DNA was analyzed by agarose gel electrophoresis.

3.4 Agarose Gel Electrophoresis

Preparation of sample for running gel: 2 ul 6 X loading dye was added to 1 0 ul reaction mix. Electrophoresis : 0.8% agarose gel (0.6-1.2 % depends on the size of DNA). Every 0.8 g agarose was add 100 ml 1 X TBE (or TAE) buffer. The mixture was microwaved till the agarose melt complete and then cooled to 50^oC. While agarose solution was cooling, the gel box and comb was assembled and then the gel was poured into it. After the gel was solidified, the sample was loaded and run with 50-150V. After the DNA dye was on appropriate site, the gel was stained in EtBr 10 ug/ml for 5 minutes and then destained in running water for 10-15 minutes. The gel was take picture under UV box.

3.5 PCR reaction

The primers used in this study were designed from NCBI database for amplifying target genes. The total volume of reaction mixture was 50μl, contained DNA template(10~20 ng), 5μl of 10× reaction buffer, 1μl of each 5μM primer of pairs, 4μl of 2.5μM dNTP, 0.5μl of Tag (5U/μl) and fulfilled with ddH2O. The PCR reaction was as following :

Step 1 : 95^oC for 10 minutes Step 2 : 95^oC for 1 minutes Step 3 : 50~60^oC for 1 minutes Step 4 : 72^oC for 1 minutes

Step 5 : back to step 2 and repeat for 29 cycles Step 6 : 72^oC for 5 minutes

After the PCR reaction was finished, the PCR products were analyzed by agarose gel electrophoresis.

3.6 PCR clean up

For removing the extra material from PCR reaction mixtures, the PCR products were clean up with ExcelPure^TM PCR Cleanup kit (Cat. No.CU250).

3.7 Gel extraction

For isolating DNA from agarose gel, the band was excised out of the agarose gel with PCR clean-up/Gel Extraction kit (Premier, N-DCE050).

3.8 Dephosphorylation

Restriction-digested vector (restriction enzyme and buffer removed, DNA in water or TE buffer) was incubated with Shrimp Alkaline Phosphatase (1 unit/μg DNA) (Promega, Cat. No.M8201) at 37°C for 15 minutes in 1X SAP reaction buffer in a final volume of 30–50μl. This is a sufficient amount of SAP to completely dephosphorylate the vector regardless of overhang type (5´, 3´ or blunt). Inactivate SAP by heating to 65°C for 15 minutes.

3.9 DNA ligation

For ligation of vector DNA and insert DNA, the total reaction volume was 10~30μl. The reaction solution contained 1 unit T4 DNA ligase, 1~3μl of 10× ligase buffer, vector DNA and insert DNA (the ratio of vector DNA to insert DNA was 1:3)

3.10 Yeast transformation

S. cerevisiae cells were grown in 10 ml of YPD or selective medium overnight at 30 for 18 hours with 300 rpm (OD℃ 600 > 1.0). 1 ml of culture was transfered into eppendorf tubes (If more cells needed, use 2 ml of overnight culture, especially, when selective medium is used). Cells was spun for 30 seconds - 1 minutesute at high speed , and poured off supernatant. Cells was resuspended with 1 ml of 0.1 M of LioAC/TE, then spun for 30 secconds - 1 minutesute at low speed and poured off supernatant, repeated twice. Then cells was resuspended gently with 0.1 ml of 0.1 M of LioAC/TE. 0.1~1 μg plasmid (to be transformed) and 3 ~10 μl of boiled 10 mg/ml salmon sperm DNA as carrier (DNA was boiled for 1 minutesute and kept on ice right away) were added. The reaction mixture were mixed gently, then 0.4 ml of 50%

PEG3350 /0.1 M of LioAC/TE was added. The reaction was then incubated at 30 on ℃ wheel for 30 minutes or sit in 30 incubator for 30℃ -60 minutes. The reaction mixture was then subjected to heat shock at 42 for 20 ℃ minutes in water bath. Cells were spun very gently at low speed for 30 seconds, and the supernatant was discarded. The reaction was further spun for 5 seconds, then all supernatant was removed with 200 μl pipette tip. 150 μl of SD or TE was added to resuspend cells. Then the cells were plated on selective media (SD agar/Uridine,Histidine) and incubated at 30 ℃ incubator for 3-5 days.

3.11 Western blot analysis

Protein sample from cell extract : S. cerevisiae cells were grown in 5 ml of SD broth/Uridine,Histidine at 30 for 48 hours wi℃ th 300 rpm (OD600 ~ 2.0). The cell solution was centrifuged at 2500 rpm at 4 for 5 ℃ minutes. After the supernatant was

discarded, the cells was resuspended in 10 ml Z buffer and then centriguged at 2500 rpm at 4 for 5 ℃ minutes once again. The cells was resuspended in 200μl breaking buffer in the eppendorf, and added 12.5μl PMSF and glass beads (near the fluid surface). The mixture was vortex at 4℃for 15 seconds, repeated four times. After the cells was broken completely, 200μl breaking buffer was added and vortex for 10 seconds. The cell mixture was centrifuged at 4 , 13000 rpm for 15 ℃ minutes. The supernatant was transferred to cooled eppendorf and added the same volume of 2×

SDS-PAGE loading dye. The protein sample was stored at -20 .℃

Protein samples from supernatant of 5 ml cultured media was obtained as following : S. cerevisiae cells were grown in 5 ml of SD broth/Uridine,Histidine at 30 for 48 hours with 300 rpm (OD℃ 600 ~ 2.0) . The cell solution was centrifuged at 2500 rpm at 4 for 5 ℃ minutes. Then the supernatant was transferred to 15ml tube or concentrated by Amicon Ultra-15 (Cat. No.UFC9 010 08). Equal amount of the protein sample was added the same volume of 2× SDS-PAGE loading dye and stored at -20 .℃

Protein samples from supernatant of 50 ml cultured media was obtained as following: S. cerevisiae cells were grown in 5 ml of SD broth/Uridine,Histidine at 30 for 48 hours with 300 rpm (OD℃ 600 ~ 2.0). Then 2ml of cell solution was transferred to 50 ml of SD broth/Uridine,Histidine and incubated at 30℃ for 48 hours (OD600 ~ 2.0). The cell solution was centrifuged at 2500 rpm at 4℃ for 5 minutes.

The supernatant was concentrated by Amicon Ultra-15 (Cat. No.UFC9 010 08). The protein sample was added the same volume of 2× SDS-PAGE loading dye and stored at -20 .℃

3.11.2 SDS-PAGE Electrophoresis

thin spacer was used and a comb was chosen--number of wells varies. Then APS and TEMED was added to resolving gel and mixed. The resolving gel mixture was poured into the spacer about 3-3.5 ml per gel and kept in room temperature for polymerizing.

After resolving gel polymerizes, APS and TEMED were added to stacking gel mixture and poured gently on top of the resolving gel. Then the comb was set up to solidify. After the gel was solidified, the comb was removed and the gel was placed in holder/electrode and then was transferred to running tank. The tank was filled with 1X Running Buffer ( the inside and outside buffer chambers was kept separated).

10~50μl of protein samples were loaded in each well. The electrophoresis was conducted at 100V through the stacking gel, and then at 100V-200V until the dye front reached the bottom of the gel.

3.11.3 Western blot

After preparation of samples and SDS-PAGE electrophoresis, the gel was incubated in about 20 ml of transfer buffer for 15 minutes with shaking. In the mean time, one piece of Nitrocellulose membrane for each gel was incubated in methanol for 1 minute. After that, methanol was replaced with transfer buffer. The Nitrocellulose membrane was placed on top of the gel, and one filter paper placed on top of that and two filter papers below. of that. The proteins were transferred to Nitrocellulose membrane at 0.09 A for 37 minutes by TRANS-BLOT® SD CELL 221BR (BIO-RAD) for a gel. Non-specific binding sites were blocked by incubation of Nitrocellulose membrane in 20 ml of blocking buffer for 60 minutes at room temperature with continuous, gentle rotary agitation. Nitrocellulose membrane was then incubated with a dilution 1:1000 primary antibody-HRP conjugated (in blocking buffer) for 3 hours at room temperature, with continuous gentle agitation, or for 16 hours overnight at 4 °C. Nitrocellulose membrane was rinsed in 10 ml of TBST twice

and was washed in 20 ml of TBST for 1 x 20 minutes followed by 4 x 5 minutes, with continuous, rotary agitation on each occasion. Again, agitation should be vigorous but not too much so as to damage the membrane. The peroxidase detection method was applied. The membrane was incubated with HRP substrate and covered with saran wrap after removing the excessive solution from the surface. Then it was exposed to X-ray film in dark room for proper exposure time and treated with developer and fixer solution.

3.12 Fluorescence staining

S. cerevisiae cells were grown in 5 ml of SD broth/Uridine,Histidine at 30℃ for 48 hours with 300 rpm (OD600 ~ 2.0). For fixing, 50μl of 36.5% formaldehyde was added to 316 μl of cell solution and the mixture was incubated at room temperature for 15 minutes. Then the cells were centrifuged at 13000 rpm for 5 minutes. The supernatant was removed with 200μl pipette, and the cells were washed with 500μl of PBS solution. Then the cells were centrifuged at 13000 rpm for 5 minutes and washed once again. The cells were resuspended in 100 μl of lyticase digestion solution at 30 for 30 ℃ minutes and then added 900 μl PBS. Then the cells were centrifuged at 4 , 2000 rpm for 5 ℃ minutes. The supernatant was removed with 200μl pipette, and the cells were washed with 500μl of PBS solution. Then the cells were centrifuged a 4 , 2000 rpm for 5 ℃ minutes and wash once again. The cells were resuspended in 200 μl PBS. 10 μl of cell solution was placed on the polylysine coated slide at room temperature for 5~10 minutes. Then the slide was washed with 4℃ PBS and dry at room temperature.

For membrane labeling, the slide coated cells was added 10 μl of 1 μM Alexa Fluor 594 WGA for 5 minutes. Then the slide was washed with PBS to remove excessive dye and dry the slide. After the slide was dry, 75% glycerol was added on the slide and the cover glass was covered on it. The slide was sealed with nail polish.

Then the samples were viewed with a fluorescence microscope or confocalmicroscope with a standard fluorescent filter. For cell wall labeling, the procedure was the same as above, but the cells were stained with 10 μl of Calcofluor white without litycase treating.

IV. Result

In this study, the CaENO1 gene of C. albicans was cloned into YEP363 plasmid (named YEP363-CaENO1-TP) and expressed in S. cerevisiae (10560-2B). In order to examine whether the protein was secreted, I modified the CaENO1gene with HAHIS and EGFP tags fused to 3’ end.

4.1 The CaENO1 gene was expressed in S. cerevisiae

4.1.1 The restriction enzyme map of the YEP363-CaENO1-TP plasmid

The YEP363-CaENO1-TP plasmid was made previously in the laboratory. It was diagramed into a circular map about 10 kb (shown in the Figure 2A ) and composed of the CaENO1 gene tagged with HA3HIS6 tag, pACT1 promoter, Leu2 gene, lacZ gene, ampR (ampicillin resistance), and 2μ (origin of S. cerevisiae). For selecting the correct plasmid, the possible samples were subjected to Nde I restriction digestion.

Accordingly, three DNA fragments of 1868 bp, 3022 bp and 5632 bp will be produced, as those in the band A, band B and band C in lane1-6 of Figure 2B. Lane 7 was undigested plasmid. The plasmid samples from lane 1-6 were in accord with the prediction and used for following experiments.

4.1.2 The expression of CaENO1 gene

The YEP363-CaENO1-TP plasmid was transformed into strain 10560-2B to express CaENO1 gene, and this transformed strain was named 10560-2B-f1 in this study. The protein product of the CaENO1 gene tagged HA3HIS6 was about 53 kDa, named eno-tp (tp, tag protein HA3HIS6) in this study. To ensure the strain 10560-2B-f1 can express target protein eno-tp, I incubated the cells at 30 for 48 ℃

hours and broke them to extract the proteins. Then the protein sample was checked with western blot. If the protein was expressed in the cell, a signal in the protein sample will be detected at about 53 kDa by anti-HA-HRP antibody with western blot.

In the western blot result shown in Figure 3, a signal correspond to the size of the protein sample was detected (band A near 55kDa in lane 1-4) and the positive control (E-tag, E-protein tagged with HAHIS tag was about 40 kDa) and negative control (the strain transformed YEP363 was named 10560-2B-f0 in this study) were in lane 5 and lane 6, respectively. According to this result, the protein eno-tp was in accord with the prediction. The positive control (E-tag) was extracted from E. coli, and it was detected at about 55 kDa (band A in lane 5) lager than the prediction in this experiment. It might be result from incompletely denaturing.

4.1.3 The secretion of the eno-tp

In order to detect the secreted eno-tp, the centrifugation of cultured medium was collected and checked with western blot. If the protein was secreted outside the cell, a signal about 53 kDa would be detected in the medium by anti-His-HRP antibody with western analysis. In the western blot result shown in Figure 4, a signal was detected in the medium sample by anti-HA-HRP antibody (as shown in the band B’ in lane 3-6) at about 55 kDa. According to this result, the eno-tp can be secreted outside the S.

cerevisiae cell. The positive control (E-tag, E-protein tagged with HAHIS tag was about 40 kDa) and the negative control was shown in lane 2 and lane 1, respectively.

The positive control (E-tag) was detected at about 40 kDa ( the band A in lane 2 ) in accord with prediction, but also detected at about 55 kDa and 80 kDa (the band B and C in lane 2) larger than the prediction in this experiment. It might be result from the incompletely denaturing.

4.2 The analysis of CaENO1 -EGFP fusion protein

Because the eno-tp protein cannot be observed for its localization inside the cell, CaENO1 gene was further tagged with Enhanced Green Fluorescent Proteins (EGFP) for visualization in this study. The EGFP is a 29 kDaa recombinant protein with Ex/Em = 488/507 nm, and it is a powerful tool for the visualization of tagged proteins.

4.2.1 The construction of the expressed plasmid

For the construction, the EGFP gene was amplified by PCR from pEGFP-N2 plasmid. Two kinds of PCR products (XbaΙ-EGFP-XbaΙ and XbaΙ-EGFP-PstΙ) were obtained through two pairs of primers (EGFP-L-XbaⅠSpeⅠ, EGFP-R-XbaⅠ and EGFP-L-Xba ⅠSpe Ⅰ, EGFP-R- Pst Ⅰ) by PCR. The PCR products of XbaΙ-EGFP-XbaΙ was predicted to be 739 bp (the band A in lane 1of Figure 5B) and Xba1-EGFP-Pst1 was predicted to be 742 bp, (the band B in lane 2 of Figure 5B).

And then the PCR products as insert were ligated into vector (YEP363-CaENO1-TP plasmid) in frame to form recombinant plasmids. The diagram of construct was as shown in Figure 5A, and one of the construct was tagged with HA3HIS6 and the other not, named YEP363-CaENO1-EGFP-TP and YEP363-CaENO1-EGFP respectively in this study. The recombinant plasmids were checked by restriction enzyme digestion.

The plasmid YEP363-CaENO1-EGFP was about 11115 bp and would be digested into four DNA fragments 721 bp, 929 bp , 5694 bp and 3771 bp ( the band a, b, c and d in lane1 of Figure 6B ) by XbaΙ and BsrGI. The plasmid YEP363-CaENO1-EGFP-TP was about 11251 bp and would be digested into four DNA fragments 721 bp, 929 bp, 3771 bp and 5830 bp DNA fragments ( the band a, b, c and d in lane1 of Figure 6C ) by XbaΙ and BsrGI. Both of them were in accord with the prediction. The recombinant

eno-EGFP-tp and eno-EGFPp (named in this study).

The S. cerevisiae strain transformed with the YEP363-CaENO1-EGFP-TP plasmid and YEP363-CaENO1-EGFP plasmid was named 10560-2B-f2 and 10560-2B-f3 in this study.

4.2.2 The expression of CaENO1-EGFP fusion protein

To ensure the strain 10560-2B-f2 and 10560-2B-f3 can express target fusion protein, eno-EGFP-tp and eno-EGFPp, the cells were incubated at 30 for 48 hours ℃ and broken to extract the protein sample. Then the protein sample was analyzed with western blot. The molecular weight of eno-EGFP-tp and eno-EGFPp was predicted to be about 79 kDa and 75kDa. If the fusion protein was expressed in the cell, the protein sample would be detected by antibody with western blot. In this experiment, two kinds of antibody (anti-HA-HRP antibody and anti-EGFP antibody) was used.

The result with the anti-HA-HRP antibody was shown in Figure 7A. The positive control E-tag (the band A in lane 1) and the negative control was shown in lane 1 and lane 2, respectively. Tow signal in the sample of the eno-EGFP-tp was detected at about 79kDa and 72 kDa (band C and D in lane 4) and there was no signal for eno-EGFPp (lane 3). In addition, the result used anti-EGFP antibody was shown in Figure 7B, the eno-EGFPp was detected at about 75kDa (band E) in lane 2 and eno-EGFP-tp about 79kDa (band F) in lane 3. It follows that eno-EGFP-tp and eno-EGFPp were expressed in the cells.

4.2.3 The secretion of the CaENO1-EGFP fusion protein

In order to examine whether the EGFP fusion protein could be secreted outside the S. cerevisiae cell, the centrifugate of cultured medium was collected and checked with western blot. If the protein was secreted outside the cell, the medium sample would be

detected by antibody with western blot. In this experiment, two kinds of antibodies (anti-HA-HRP antibody and anti-EGFP antibody) were used.

In Figure 8A (anti-HA-HRP antibody used), the eno-tp was detected at about 55kDa (band B in lane 2 ) and the eno-EGFP-tp was detected at about 79kDa (band C in lane 3); the positive control E-tag was about 40 kDa (band A in lane 1) and the negative control was in lane 2. In Figure 8B (anti-EGFP-HRP antibody used), the eno-EGFPp was detected at about 75kDa (band D in lane 2) and the eno-EGFP-tp can not be detected. Negative control was in lane 1.

According to the result, both eno-EGFP-tp and eno-EGFPp can be detected in the culture medium.

4.2.4 The fluorescence observation of CaENO1-EGFP fusion protein

To further examine whether the EGFP fusion protein could be expressed, I incubated the transformed cells at 30 for 48 hours℃ and observed under fluorescence microscope with 400X magnification.

As shown in Figure 9, 10560-2B-f2 (shown in B) and 10560-2B-f3(shown in C) were fluorescent under fluorescence microscope. 10560-2B-f0 (shown in A) was negative control in this data. It follows that the EGFP fusion protein was fluorescent.

4.3 The analysis of truncated CaENO1

In this experiment, in order to determine which region of CaENO1 gene was critical to secretion, I designed different constructs of truncated-CaENO1 fused to EGFP and compared their activities to the control construct. The diagram of this experiment was shown in Figure 10.

The fragments of truncated CaENO1 were amplified by PCR. I designed several pairs of primers for PCR amplification of different length of truncated CaENO1. The

The fragments of truncated CaENO1 were amplified by PCR. I designed several pairs of primers for PCR amplification of different length of truncated CaENO1. The