Result

Fig. 1 Immunofluorescence labeling of transformed cells: green fluorescence corresponds to cells labeled with Mouse Anti HA-Tag Polyclonal antibody and (Fluorescein (FITC)–conjugated Affinipure Goat Anti-Mouse IgG(H+L), and red fluorescence corresponds to cells labeled with Rabbit Anti Flag-Tag Polyclonal antibody and Goat Anti Rabbit RPE Polyclonal antibody.

Immunofluorescence analysis of β-glucosidase surface display Pichia pastoris using flow cytometry.

We fused HA-tag with N-terminal of bglX gene on ZXFα-bglX vector. Label cells with Mouse Anti HA-Tag Polyclonal antibody and Fluorescein (FITC)–conjugated Affinipure Goat Anti-Mouse IgG(H+L). Result of flow cytometry analysis is as follows: fluorescence intensity x-mean value of Pichia pastoris with pPICZα empty vector is 0.306, with bglX-ZXFα is 23.4.

Fig. 2-1 Left: pPICZα negative control (x-mean: 0.306/1.17); Right: bglX (x-mean: 23.4/23.6)

Immunofluorescence analysis of endoxylanase surface display Pichia pastoris using flow cytometry.

We fused HA-tag with N-terminal of xyn gene on ZXFα-xyn vector. Label cells with Mouse Anti HA-Tag Polyclonal antibody and Fluorescein (FITC)–conjugated Affinipure Goat Anti-Mouse IgG(H+L). Result of flow cytometry analysis is as follows: fluorescence intensity x-mean value of Pichia pastoris with pPICZα empty vector is 0.62, with xyn-ZXFα is 67.

Fig. 2-2 Left: pPICZα negative control (x-mean: 0.62/4.31); Right: xyn (x-mean: 67/67.6)

Immunofluorescence analysis of β-xylosidase Ruxyn surface display Pichia pastoris using flow cytometry.

We fused FLAG-tag with N-terminal of Ruxyn gene on Ruxyn-pPIC9k vector. Label cells with Rabbit Anti Flag-Tag Polyclonal antibody and Goat Anti Rabbit RPE Polyclonal antibody. Result of flow cytometry analysis is as follows: fluorescence intensity x-mean value of Pichia pastoris with pPIC9k empty vector is 1.14, with Ruxyn-pPIC9k is 8.56.

Fig. 2-3 Left: pPIC9k negative control (x-mean: 1.14/13.4); Right: Ruxyn (x-mean: 8.56/19.9)

Immunofluorescence analysis of endoglucanase cel5A surface display Pichia pastoris using flow cytometry.

We fused FLAG-tag with N-terminal of cel5A gene on cel5A-pPIC9k vector. Label cells with Rabbit Anti Flag-Tag Polyclonal antibody and Goat Anti Rabbit RPE Polyclonal antibody. Result of flow cytometry analysis is as follows: fluorescence intensity x-mean value of Pichia pastoris with pPIC9k empty vector is 1.14, with cel5A-pPIC9k is 5.55.

Fig. 2-4 Left: pPIC9k negative control (x-mean: 1.14/13.4); Right: cel5A (x-mean: 5.55/16.5)

Construction of endoglucanase cel5A display vector

Figure 3-1 Agarose gel electrophoresis analysis of endoglucanase cel5A display vector colony PCR.
1: negative control; 2: positive control; 3: cel5A1; 4: cel5A2 5: cel5A3
Figure 3-2 Agarose gel electrophoresis analysis of double digestion of endoglucanase cel5A by EcoRI and MluI.
1: cel5A1; 2: cel5A2

Construction of β-glucosidase bglX display vector

Figure 4-1 Agarose gel electrophoresis analysis of β-glucosidase bglX-pMD18-T colony PCR.
1: bglX-pMD18-T1; 2: bglX-pMD18-T2; 3: bglX-pMD18-T3; 4: bglX-pMD18-T4; 5: bglX-pMD18-T5; 6: bglX-pMD18-T6; 7: bglX-pMD18-T7; 8: bglX-pMD18-T8; 9: negative control; 10: positive control

Figure 4-2 Agarose gel electrophoresis analysis of double digestion of β-glucosidase bglX-pMD18-T by NheI and XhoI.
1: bglX-pMD18-T1; 2: bglX-pMD18-T2; 3: bglX-pMD18-T3; 4: bglX-pMD18-T4; 5: bglX-pMD18-T5; 6: bglX-pMD18-T6; 7: Ruxyn-pMD18-T1; 8: Ruxyn-pMD18-T2; 9: Ruxyn-pMD18-T3; 10: xyn-pMD18-T1; 11: xyn-pMD18-T2; 12: xyn-pMD18-T3
Since bglX gene (2280bp) and pMD18-T vector (2692bp) have similar lengths, it is difficult to discriminate the two from the result.

Figure 4-3 Agarose gel electrophoresis analysis of β-glucosidase bglX-pPICZXFα colony PCR.
1: bglX-pPICZXFα1; 2: bglX-pPICZXFα2; 3: bglX-pPICZXFα3; 4: positive control; 5: negative control

Figure 4-4 Agarose gel electrophoresis of double digestion of β-glucosidase bglX-pPICZXFα using NheI and XhoI.
1: bglX-pPICZXFα1; 2: bglX-pPICZXFα2; 3: bglX-pPICZXFα3

Construction of xylanase xyn display vector

Figure 5-1 Agarose gel electrophoresis analysis of endoxylanase xyn-pMD18-T colony PCR.
1: Ruxyn-pMD18-T1; 2: Ruxyn-pMD18-T2; 3: Ruxyn-pMD18-T3; 4: Ruxyn-pMD18-T4; 5: Ruxyn-pMD18-T5; 6: Ruxyn-pMD18-T6; 7: Ruxyn-pMD18-T7; 8: Ruxyn-pMD18-T8; 9: negative control; 10: positive control

Figure 5-2 Agarose gel electrophoresis analysis of double digestion of endoxylanase xyn-pMD18-T by NheI and XhoI.
1: bglX-pMD18-T1; 2: bglX-pMD18-T2; 3: bglX-pMD18-T3; 4: bglX-pMD18-T4; 5: bglX-pMD18-T5; 6: bglX-pMD18-T6; 7: Ruxyn-pMD18-T1; 8: Ruxyn-pMD18-T2; 9: Ruxyn-pMD18-T3; 10: xyn-pMD18-T1; 11: xyn-pMD18-T2; 12: xyn-pMD18-T3

Figure 5-3 Agarose gel electrophoresis analysis of endoxylanase xyn-pPICZXFα colony PCR.
1: negative control; 2: positive control; 3: xyn-pPICZXFα3; 4: xyn-pPICZXFα2; 5: xyn-pPICZXFα1

Figure 5-4 Agarose gel electrophoresis analysis of double digestion of endoxylanase xyn-pPICZXFα by NheI and XhoI.
1: bglX-pPICZXFα1; 2: bglX-pPICZXFα2; 3: bglX-pPICZXFα3; 4: Ruxyn-pPIC9k1; 5: xyn-pMD18-T1; 6: xyn-pMD18-T2; 7: xyn-pMD18-T3