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

Construction of β-xylosidase Ruxyn display vector plasmid

Figure 6-1 Agarose gel electrophoresis analysis of β-xylosidase Ruxyn-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 6-2 Agarose gel electrophoresis analysis of double digestion of β-xylosidase Ruxyn-pMD18-T by EcoRI and MluI.
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 6-3 Agarose gel electrophoresis analysis of β-xylosidase Ruxyn-pPIC9k colony PCR.
1: negative control; 2: positive control; 3: xyn-pPICZXFα3; 4: xyn-pPICZXFα2; 5: xyn-pPICZXFα1; 6: negative control; 7: positive control; 8: Ruxyn-pPIC9k3; 9: Ruxyn-pPIC9k2; 10: Ruxyn-pPIC9k1

Figure 6-4 Agarose gel electrophoresis analysis of double digestion of β-xylosidase Ruxyn-pPIC9k by EcoRI and MluI.
1: Ruxyn-pPIC9k1

Standardization of Pichia pastoris secretory vector pPICZα

Figure 7-1 Agarose gel electrophoresis analysis of standardized secretory vector pPICZα colony PCR.
1: negative control; 2: positive control; 3: cel5A-pPICZα6; 4: cel5A-pPICZα5; 5: cel5A-pPICZα4; 6: cel5A-pPICZα3; 7: cel5A-pPICZα2; 8: cel5A-pPICZα1

Figure 7-2 Agarose gel electrophoresis analysis of double digestion of standardized secretory vector pPICZα by EcoRI and PstI.
1: cel5A-pPICZα1; 2: cel5A-pPICZα2; 3: cel5A-pPICZα3

Construction of Lignin peroxidase(LiP) BBa_K500000, Mn peroxidase(MnP) BBa_K500001, laccase BBa_K500002, Glyoxal oxidase(GLOX) BBa_K500003 secretory vectors

Figure 8-1 Agarose gel electrophoresis analysis of LiP BBa_K500000 and MnP BBa_K500001 secretory vectors colony PCR.
1: BBa_K500000-1; 2: BBa_K500000-2; 3: BBa_K500000-3; 4: BBa_K500000-4; 5: BBa_K500000-5; 6: BBa_K500000-6; 7: BBa_K500000-7; 8: BBa_K500000-8; 9: BBa_K500000-9; 10: BBa_K500000-10; 11: BBa_K500001-1; 12: BBa_K500001-2; 13: BBa_K500001-3; 14: BBa_K500001-4; 15: BBa_K500001-5; 16: BBa_K500001-6; 17: BBa_K500001-7; 18: BBa_K500001-8; 19: BBa_K500001-9; 20: BBa_K500001-10; 21: BBa_K500001-11; 22: negative control; 23: positive control

Figure 8-2 Agarose gel electrophoresis analysis of GLOX BBa_K500003 secretory vector colony PCR.
1: BBa_K500003-1; 2: BBa_K500003-2; 3: BBa_K500003-3; 4: BBa_K500003-4; 5: BBa_K500003-5; 6: BBa_K500003-6; 7: BBa_K500003-7; 8: positive control; 9: negative control

Figure 8-3 Agarose gel electrophoresis analysis of double digestion of LiP BBa_K500000 and MnP BBa_K500001 secretory vectors by EcoRI and PstI.
1: BBa_K500000-1; 2: BBa_K500000-2; 3: BBa_K500000-3; 4: BBa_K500000-4; 5: BBa_K500001-1; 6: BBa_K500001-2; 7: BBa_K500001-3; 8: BBa_K500001-4

Figure 8-4 Agarose gel electrophoresis analysis of double digestion of GLOX BBa_K500003 secretory vector by EcoRI and PstI.
1: BBa_K500003-1; 2: BBa_K500003-2; 3: BBa_K500003-3; 4: BBa_K500003-4

Point mutation and standardization of xyn

Figure 9-1 Agarose gel electrophoresis analysis of point mutation of xyn and standardized xyn-pMD18-T colony PCR.
1: negative control; 2: xyn-pMD18-T1; 3: xyn-pMD18-T2; 4: xyn-pMD18-T3; 5: xyn-pMD18-T4; 6: xyn-pMD18-T5; 7: xyn-pMD18-T6; 8: xyn-pMD18-T7; 9: xyn-pMD18-T8; 10: positive control
Figure 9-2 Agarose gel electrophoresis analysis of point mutation of xyn and double digestion of standardized xyn-pMD18-T by EcoRI and PstI.
1: xyn-pMD18-T7; 2: xyn-pMD18-T6; 3: xyn-pMD18-T5; 4: xyn-pMD18-T4; 5: xyn-pMD18-T3; 6: xyn-pMD18-T2; 7: xyn-pMD18-T1

Figure 9-3 Agarose gel electrophoresis analysis of point mutation of xyn and standardized xyn-pSB1C3 colony PCR.
1: negative control; 2: positive control; 3: xyn-pSB1C310; 4: xyn-pSB1C39; 5: xyn-pSB1C38; 6: xyn-pSB1C37; 7: xyn-pSB1C36; 8: xyn-pSB1C35; 9: xyn-pSB1C34; 10: xyn-pSB1C33; 11: xyn-pSB1C32; 12: xyn-pSB1C31

Figure 10 Sequencing result analysis of point mutation and standardization of xyn. A=>G