Team:SDSZ-China/Parts
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parts（表）
basic parts
-7alpha-HSDH
-7beta-HSDH
-LDH
-GDH
composite parts
-ChBD
--ChBD--7alpha-HSDH
--ChBD--7beta-HSDH
--ChBD-LDH
--ChBD-GDH
--ChBD-GFP
-CBD
--CBD--7alpha-HSDH
--CBD--7beta-HSDH
--CBD-LDH
--CBD-GDH
--CBD-GFP
contribution
-chbd
-cbd
-7alpha-HSDH
7alpha-HSDH (7alpha-hydroxysteroid dehydrogenase) catalyzes the oxidation of hydroxysteroids at C-7 position and back, as it converts NAD+ to NADH and back. It catalyzes the oxidation of CDCA into intermediate product 7-oxo-LAC (7-ketolithocholic acid), where the hydroxyl at C-7 position becomes carbonyl.
Source
The 7alpha-HSDH sequence is cloned from the genome of E.coli DH5alpha through PCR amplification, using the primers designed and synthesized based on its sequence.
-7beta-HSDH
7beta-HSDH (7beta-hydroxysteroid dehydrogenase) catalyzes the reduction of hydroxysteroids at C-7 position and back, as it converts NADPH to NDAP+ and back. It catalyzes the reduction of the intermediate product 7-oxo-LAC (7-ketolithocholic acid) into UDCA, the final product, where the carbonyl at C-7 position becomes hydroxyl.
Source
The 7beta-HSDH DNA is originally from the genome of R. torques ATCC 35915, and is artificially synthesized based on the sequence retrieved from the GenBank.
-LDH
LDH (lactate dehydrogenase) catalyzes the conversion of lactate to pyruvic acid and back, as it converts NAD+ to NADH and back. It is used to catalyze the reduction of pyruvate into lactate, with the presence of NADH, which is oxidized into NAD+ at the same time.
Source
The LDH DNA is originally from the genome of Lactobacillus delbrueckii subsp. Bulgaricus, and is artificially synthesized based on the sequence retrieved from the GenBank.
-GDH
GDH (glucose dehydrogenase) catalyzes the conversion of beta-D-glucose to D-glucono-1,5-lactone and back, as it converts NADP+ to NADPH and back. It is used to catalyze the oxidation of beta-D-glucose into D-glucono-1,5-lactone and reduce NADP+ into NADPH during the process.
Source
The GDH sequence is cloned from the genome of Bacillus subtilis through PCR amplification, using the primers designed and synthesized based on its sequence.
-GFP
Green fluorescent protein with his-tag.
Source
The GFP DNA is artificially synthesized based on the sequence retrieved from the GenBank.
composite parts
-ChBD
--ChBD--7alpha-HSDH
ChBD--7alpha-HSDH (T7 promoter--lac operator--RBS--His-tag--7alpha-HSDH--ChBD--T7 terminator)
This device codes for the 7alpha-HSDH--ChBD fusion protein.
Construct
The vector of 7alpha-HSDH--ChBD for its expression is pET-28x. It is formed by modifying the restriction enzyme sites EcoR I and Xba I of vector pET-28a.
The 7alpha-HSDH sequence is cloned from the genome of E.coli DH5alpha through PCR amplification, using the primers designed and synthesized based on its sequence. The restriction site BamH I is added to the upstream primer, and Hind III is added to the downstream primer.
The ChBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The ChBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind III added to the upstream primer, and Xhol I added to the downstream primer.
Firstly, the 7alpha-HSDH gene is inserted into the modified pET-28x at BamH I and Hind III, and ChBD at Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac operator--RBS--His-tag--7alpha-HSDH--ChBD--T7 terminator, is retrieved from this plasmid by PCR amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I and Pst I.
[[file:psb1c3-chbd-7a.jpeg|300px]]
[Fig. 1. pSB1C3--ChBD--7alpha-HSDH]
Usage and Biology
7alpha-HSDH(7alpha-hydroxysteroid dehydrogenase) catalyzes the oxidation of hydroxysteroids at C-7 position and back, as it converts NAD+ to NADH and back. It catalyzes the oxidation of CDCA into intermediate product 7-oxo-LAC (7-ketolithocholic acid), where the hydroxyl at C-7 position becomes carbonyl.[1]
ChBD (chitin binding domain) is able to bind to chitin. When connected to 7alpha-HSDH, ChBD is able to immobilize the enzyme 7alpha-HSDH after expression, by binding to chitin powder inside the solution.[2]
The function of chitin binding domain
The function of ChBD is tested by connecting ChBD gene with GFP gene in pET28x. The GFP-ChBD fusion protein is expressed and mixed with chitin powder. Tube 3 contains GFP-ChBD, with a significant green color in the solution. After adding chitin powder inside the tube, the color in the solution disappears and shows on the chitin powder, as presented in tube 2. It proves that the ChBD successfully binds the GFP onto the chitin powder. In contrast, the color of the solution is not changed when adding chitin powder into the GFP solution without ChBD. As a result, the ChBD is well functioned.
[[file:chbd.jpeg|250px]]
[Fig.2. ChBD function: tube1, GFP and chitin; tube2, GFP-ChBD and chitin; tube3, GFP-ChBD; tube4, water]
Expression and Immobilization
The constructed pET28x--7alpha-HSDH--ChBD plasmid is transformed into BL21(DE3) E.coli for expression. After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is broken by ultrasonication.
The resulted bacteria solution is diluted to a certain concentration and mixed thoroughly with chitin powder. As a result, the ChBD protein binds to the chitin powder, and the enzyme is successfully immobilized.
Reference
[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process Biochemistry, Elsevier, 2015.
[2] Takahisa Ikegami, Terumasa Okada, Masayuki Hashimoto, Shizuka Seino, Takeshi Watanabe, and Masahiro Shirakawa: Solution Structure of the Chitin-binding Domain of Bacillus circulans WL-12 Chitinase A1. The Journal Of Biological Chemistry, 2000.
--ChBD--7beta-HSDH
ChBD--7beta-HSDH (T7 promoter--lac operator--RBS--His-tag--7beta-HSDH--ChBD--T7 terminator)
This device codes for the 7beta-HSDH--ChBD fusion protein.
Construct
The vector of 7beta-HSDH--ChBD for its expression is pET-28x. It is formed by modifying the restriction enzyme sites EcoR I and Xba I of vector pET-28a.
The 7beta-HSDH sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The 7beta-HSDH gene is then cloned from the plasmid by PCR amplification. The restriction site BamH I is added to the upstream primer, and Hind III is added to the downstream primer.
The ChBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The ChBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind III added to the upstream primer, and Xhol I added to the downstream primer.
Firstly, the 7beta-HSDH gene is inserted into the modified pET-28x at BamH I and Hind III, and ChBD at Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac operator--RBS--His-tag--7beta-HSDH--ChBD--T7 terminator, is retrieved from this plasmid by PCR amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I and Pst I.
[[file:psb1c3-chbd-7b.jpeg|300px]]
[Fig. 1. pSB1C3--ChBD--7beta-HSDH]
Usage and Biology
7beta-HSDH(7beta-hydroxysteroid dehydrogenase) catalyzes the reduction of hydroxysteroids at C-7 position and back, as it converts NADPH to NDAP+ and back. It catalyzes the reduction of the intermediate product 7-oxo-LAC (7-ketolithocholic acid) into UDCA, the final product, where the carbonyl at C-7 position becomes hydroxyl.[1]
ChBD (chitin binding domain) is able to bind to chitin. When connected to 7beta-HSDH, ChBD is able to immobilize the enzyme 7beta-HSDH after expression, by binding to chitin powder inside the solution.[2]
The function of chitin binding domain
The function of ChBD is tested by connecting ChBD gene with GFP gene in pET28x. The GFP-ChBD fusion protein is expressed and mixed with chitin powder. Tube 3 contains GFP-ChBD, with a significant green color in the solution. After adding chitin powder inside the tube, the color in the solution disappears and shows on the chitin powder, as presented in tube 2. It proves that the ChBD successfully binds the GFP onto the chitin powder. In contrast, the color of the solution is not changed when adding chitin powder into the GFP solution without ChBD. As a result, the ChBD is well functioned.
[[file:chbd.jpeg|250px]]
[Fig.2. ChBD function: tube1, GFP and chitin; tube2, GFP-ChBD and chitin; tube3, GFP-ChBD; tube4, water]
Expression and Immobilization
The constructed pET28x--7beta-HSDH--ChBD plasmid is transformed into BL21(DE3) E.coli for expression. After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is broken by ultrasonication.
The resulted bacteria solution is diluted to a certain concentration and mixed thoroughly with chitin powder. As a result, the ChBD protein binds to the chitin powder, and the enzyme is successfully immobilized.
Reference
[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process Biochemistry, Elsevier, 2015.
[2] Takahisa Ikegami, Terumasa Okada, Masayuki Hashimoto, Shizuka Seino, Takeshi Watanabe, and Masahiro Shirakawa: Solution Structure of the Chitin-binding Domain of Bacillus circulans WL-12 Chitinase A1. The Journal Of Biological Chemistry, 2000.
--ChBD-LDH
ChBD-LDH (T7 promoter--lac operator--RBS--His-tag--LDH--ChBD--T7 terminator)
This device codes for the LDH-ChBD fusion protein.
Construct
The vector of LDH-ChBD for its expression is pET-28x. It is formed by modifying the restriction enzyme sites EcoR I and Xba I of vector pET-28a.
The LDH sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The LDH gene is then cloned from the plasmid by PCR amplification. The restriction site BamH I is added to the upstream primer, and Hind III is added to the downstream primer.[1]
The ChBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The ChBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind III added to the upstream primer, and Xhol I added to the downstream primer.[2]
Firstly, the LDH is inserted into the modified pET-28x at BamH I and Hind III, and ChBD at Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac operator--RBS--His-tag--LDH--ChBD--T7 terminator, is retrieved from this plasmid by PCR amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I and Pst I.
[[file:psb1c3-chbd-ldh.jpeg|300px]]
[Fig. 1. pSB1C3-ChBD-LDH]
Usage and Biology
LDH(lactate dehydrogenase) catalyzes the conversion of lactate to pyruvic acid and back, as it converts NAD+ to NADH and back. It is used to catalyze the reduction of pyruvate into lactate, with the presence of NADH, which is oxidized into NAD+ at the same time.[1]
ChBD (chitin binding domain) is able to bind to chitin. When connected to LDH, ChBD is able to immobilize the enzyme LDH after expression, by binding to chitin powder inside the solution.[2]
The function of chitin binding domain
The function of ChBD is tested by connecting ChBD gene with GFP gene in pET28x. The GFP-ChBD fusion protein is expressed and mixed with chitin powder. Tube 3 contains GFP-ChBD, with a significant green color in the solution. After adding chitin powder inside the tube, the color in the solution disappears and shows on the chitin powder, as presented in tube 2. It proves that the ChBD successfully binds the GFP onto the chitin powder. In contrast, the color of the solution is not changed when adding chitin powder into the GFP solution without ChBD. As a result, the ChBD is well functioned.
[[file:chbd.jpeg|250px]]
[Fig.2. ChBD function: tube1, GFP and chitin; tube2, GFP-ChBD and chitin; tube3, GFP-ChBD; tube4, water]
Expression and Immobilization
The constructed pET28x-LDH-ChBD plasmid is transformed into BL21(DE3) E.coli for expression. After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is broken by ultrasonication.
The resulted bacteria solution is diluted to a certain concentration and mixed thoroughly with chitin powder. As a result, the ChBD protein binds to the chitin powder, and the enzyme is successfully immobilized.
Reference
[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process Biochemistry, Elsevier, 2015.
[2] Takahisa Ikegami, Terumasa Okada, Masayuki Hashimoto, Shizuka Seino, Takeshi Watanabe, and Masahiro Shirakawa: Solution Structure of the Chitin-binding Domain of Bacillus circulans WL-12 Chitinase A1. The Journal Of Biological Chemistry, 2000.
--ChBD-GDH
ChBD-GDH (T7 promoter--lac operator--RBS--His-tag--GDH--ChBD--T7 terminator)
This device codes for the GDH-ChBD fusion protein.
Construct
The vector of GDH-ChBD for its expression is pET-28x. It is formed by modifying the restriction enzyme sites EcoR I and Xba I of vector pET-28a.
The GDH sequence is cloned from the genome of Bacillus subtilis through PCR amplification, using the primers designed and synthesized based on its sequence. The restriction site BamH I is added to the upstream primer, and Hind III is added to the downstream primer.
The ChBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The ChBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind III added to the upstream primer, and Xhol I added to the downstream primer.
Firstly, the GDH is inserted into the modified pET-28x at BamH I and Hind III, and ChBD at Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac operator--RBS--His-tag--GDH--ChBD--T7 terminator, is retrieved from this plasmid by PCR amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I and Pst I.
[[file:psb1c3-chbd-gdh.jpeg|300px]]
[Fig. 1. pSB1C3-ChBD-GDH]
Usage and Biology
GDH(glucose dehydrogenase) catalyzes the conversion of beta-D-glucose to D-glucono-1,5-lactone and back, as it converts NADP+ to NADPH and back. It is used to catalyze the oxidation of beta-D-glucose into D-glucono-1,5-lactone and reduce NADP+ into NADPH during the process.[1]
ChBD (chitin binding domain) is able to bind to chitin. When connected to GDH, ChBD is able to immobilize the enzyme GDH after expression, by binding to chitin powder inside the solution.[2]
The function of chitin binding domain
The function of ChBD is tested by connecting ChBD gene with GFP gene in pET28x. The GFP-ChBD fusion protein is expressed and mixed with chitin powder. Tube 3 contains GFP-ChBD, with a significant green color in the solution. After adding chitin powder inside the tube, the color in the solution disappears and shows on the chitin powder, as presented in tube 2. It proves that the ChBD successfully binds the GFP onto the chitin powder. In contrast, the color of the solution is not changed when adding chitin powder into the GFP solution without ChBD. As a result, the ChBD is well functioned.
[[file:chbd.jpeg|250px]]
[Fig.2. ChBD function: tube1, GFP and chitin; tube2, GFP-ChBD and chitin; tube3, GFP-ChBD; tube4, water]
Expression and Immobilization
The constructed pET28x-GDH-ChBD plasmid is transformed into BL21(DE3) E.coli for expression. After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is broken by ultrasonication.
The resulted bacteria solution is diluted to a certain concentration and mixed thoroughly with chitin powder. As a result, the ChBD protein binds to the chitin powder, and the enzyme is successfully immobilized.
Reference
[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process Biochemistry, Elsevier, 2015.
[2] Takahisa Ikegami, Terumasa Okada, Masayuki Hashimoto, Shizuka Seino, Takeshi Watanabe, and Masahiro Shirakawa: Solution Structure of the Chitin-binding Domain of Bacillus circulans WL-12 Chitinase A1. The Journal Of Biological Chemistry, 2000.
--ChBD-GFP
ChBD-GFP (T7 promoter--lac operator--RBS--His-tag--GFP--ChBD--T7 terminator)
This device codes for the GFP-ChBD fusion protein.
Construct
The vector of GFP-ChBD for its expression is pET-28x. It is formed by modifying the restriction enzyme sites EcoR I and Xba I of vector pET-28a.
The GFP sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The GFP gene is then cloned from the plasmid by PCR amplification. The restriction site BamH I is added to the upstream primer, and Hind III is added to the downstream primer.
The ChBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The ChBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind III added to the upstream primer, and Xhol I added to the downstream primer.
Firstly, the GFP is inserted into the modified pET-28x at BamH I and Hind III, and ChBD at Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac operator--RBS--His-tag--GFP--ChBD--T7 terminator, is retrieved from this plasmid by PCR amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I and Pst I.
[[file:psb1c3-chbd-gfp.jpeg|300px]]
[Fig. 1. pSB1C3-ChBD-GFP]
Usage and Biology
This part is used to test the function of ChBD (chitin binding domain), which is able to bind to chitin.[1]
Expression
The constructed pET28x-GFP-ChBD plasmid is transformed into BL21(DE3) E.coli for expression. After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is broken by ultrasonication.
Proof of the ChBD function
The resulted solution after expression is mixed with chitin powder. Also, the solution with GFP-ChBD but without chitin, solution with the presence of GFP and chitin, and water in comparison, are presented.
Tube 3 contains GFP-ChBD, with a significant green color in the solution. After adding chitin powder inside the tube, the color in the solution disappears and shows on the chitin powder, as presented in tube 2. It proves that the ChBD successfully binds the GFP onto the chitin powder. In contrast, the color of the solution is not changed when adding chitin powder into the GFP solution without ChBD. As a result, the ChBD is well functioned.
[[file:chbd.jpeg|250px]]
[Fig.2. ChBD function: tube1, GFP and chitin; tube2, GFP-ChBD and chitin; tube3, GFP-ChBD; tube4, water]
Reference
[1] Takahisa Ikegami, Terumasa Okada, Masayuki Hashimoto, Shizuka Seino, Takeshi Watanabe, and Masahiro Shirakawa: Solution Structure of the Chitin-binding Domain of Bacillus circulans WL-12 Chitinase A1. The Journal Of Biological Chemistry, 2000.
-CBD
--CBD--7alpha-HSDH
CBD--7alpha-HSDH (T7 promoter--lac operator--RBS--His-tag--7alpha-HSDH--CBD--T7 terminator)
This device codes for the 7alpha-HSDH--CBD fusion protein.
Construct
The vector of 7alpha-HSDH--CBD for its expression is pET-28x. It is formed by modifying the restriction enzyme sites EcoR I and Xba I of vector pET-28a.
The 7alpha-HSDH sequence is cloned from the genome of E.coli DH5alpha through PCR amplification, using the primers designed and synthesized based on its sequence. The restriction site BamH I is added to the upstream primer, and Hind III is added to the downstream primer.
The CBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The CBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind III added to the upstream primer, and Xhol I added to the downstream primer.
Firstly, the 7alpha-HSDH gene is inserted into the modified pET-28x at BamH I and Hind III, and CBD at Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac operator--RBS--His-tag--7alpha-HSDH--CBD--T7 terminator, is retrieved from this plasmid by PCR amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I and Pst I.
[[file:Psb1c3-cbd-7a.jpeg|300px]]
[Fig. 1. pSB1C3--CBD--7alpha-HSDH]
Usage and Biology
7alpha-HSDH(7alpha-hydroxysteroid dehydrogenase) catalyzes the oxidation of hydroxysteroids at C-7 position and back, as it converts NAD+ to NADH and back. It catalyzes the oxidation of CDCA into intermediate product 7-oxo-LAC (7-ketolithocholic acid), where the hydroxyl at C-7 position becomes carbonyl.[1]
CBD (cellulose binding domain) is able to bind to cellulose. When connected to 7alpha-HSDH, CBD is able to immobilize the enzyme 7alpha-HSDH after expression, by binding to the gauze inside the solution on its cellulose.[2]
The function of cellulose binding domain
The function of CBD is tested by connecting CBD gene with GFP gene in pET28x. The GFP-CBD fusion protein is expressed and mixed with a gauze piece. The green fluorescent on the gauze is not significantly reduced after washing, proving that the CDB is well functioned. In comparison, no green fluorescent is left after washing the gauze mixed with GFP-ChBD (Chintin binding domain).
[[file:cbd-before.jpeg|150px]]
[Fig. 2. GFP-CBD on gauze before washing]
[[file:cbd-after.jpeg|150px]]
[Fig. 3. GFP-CBD on gauze after washing]
[[file:chbd-before.jpeg|150px]]
[Fig. 4. GFP-ChBD on gauze before washing]
[[file:chbd-after.jpeg|150px]]
[Fig. 5. GFP-ChBD on gauze after washing]
Expression and Immobilization[2]
The constructed pET28x--7alpha-HSDH--CBD plasmid is transformed into BL21(DE3) E.coli for expression. After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is broken by ultrasonication.
The resulted bacteria solution is diluted to a certain concentration and mixed with gauze piece and the gauze piece is washed three times by ddH2O afterwards. As a result, the CBD protein binds to the cellulose on gauze, and the enzyme is successfully immobilized.
Enzyme Activity
The oxidation of CDCA to 7-OXO-LCA using 7alpha-HSDH
[[file:reaction1-1.jpeg|400px]]
[Fig. 6. Reaction process]
7a-HSDH from Ecoli 5a is an NAD+ dependent enzyme. The 3 mL reaction consists of 150mM phosphate buffer(pH 8.0), 10mM CDCA, 0.2mM NAD+. The reaction started when the solution is combined with 7a-HSDH-CBD-enzyme-binding gauze that in different concentration, includes 60ul, 120ul, and 180ul liquid supernatant of ultrasonication bacteria solution. The control group was testify under the same solution and condition but using pET28x-CBD liquid supernatant of ultrasonication bacteria solution to bind with gauze in the concentration of 180ul. Before adding the gauze into the solution, the gauze was washed by ddH2O for 3 times in order to purify the enzyme.
The CDCA was convert to 7OXO-LCA by loosing a pair of hydrogen(2H+and 2e-) from the 7-hydroxyl group and form 7-carbonyl group.The co-enzyme NAD+ is the acceptor of the a pair of hydrogen(2H+and 2e-) and e-, and was transformed into NADH. The enzyme activity was determined spectrophotometrically atv340 nm (ε = 6.22 mM-1 cm-1) and room temperature. One unit of activity is defined as the amount of enzyme catalyzing the synthesize of 1 mmol of NADH per min under the assay conditions used.
Result
[[file:result1-1.jpeg|400px]]
[Fig. 7. Result]
Oxidation of CDCA to 7-oxo-LCA using E. coli 7alpha-HSDH and NAD+ regeneration
[[file:reaction2-1.jpeg|400px]]
[Fig. 8. Reaction process]
CDCA was converted in a 3mL solution containing 150 mM phosphate buffer(pH 8.0), 10 mM CDCA, 30 mM sodium pyruvate, 0.25mM NAD+, combined with 3U/ml LDH and1 1 U/ml E. coli DH5a 7a-HSDH-CBD at room temperature.
The bioconversion experiments were monitored via HPLC measurements. The sample was analyzed by UV detection at 210 nm, using a mobile phase of methanol–water mixture (final ratio 80:20,pH 3.5 with phosphoric acid) using chromatographic column C18.
Result
As shown in figure, the transformation was complete after 2.5h.
[[file:hplc1-1.jpeg|500px]]
[Fig. 9. Absorbance of NADH]
[[file:hplc1-2.jpeg|400px]]
[Fig. 10. Peak area of 7oxo-LCA sample in different concentration]
[[file:hplc1-3.jpeg|500px]]
[Fig. 11. Time course]
According to the HPLC after 150minute, there is no significant increase of 7oxo-LCA, as a result, most of the CDCA has been transformed into 7oxo-LCA. And according to the HPLC the final yielding rate is 94%.
According to the Absorbance of NADH that shown in figure, the absorbance is decreased significantly after 150 minute dual to the depleted CDCA that stop the conversion of CDCA to 7oxo-LCA and NADH synthesize. Because of the abundant amount of pyruvate in the solution, the LDH that works on pyruvate still regenerate the NAD+ by taking a pair of hydrogen from NADH until most of the NADH synthesized transformed into NAD+.
Reference
[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process Biochemistry, Elsevier, 2015.
[2] Etai Shpigel, Arie Goldlust, Gilat Efroni, Amos Avraham, Adi Eshel, Mara Dekel, Oded Shoseyov: Immobilization of Recombinant Heparinase I Fused to Cellulose-Binding Domain, 1999.
--CBD--7beta-HSDH
CBD--7beta-HSDH (T7 promoter--lac operator--RBS--His-tag--7beta-HSDH--CBD--T7 terminator)
This device codes for the 7beta-HSDH--CBD fusion protein.
Construct
The vector of 7beta-HSDH--CBD for its expression is pET-28x. It is formed by modifying the restriction enzyme sites EcoR I and Xba I of vector pET-28a.
The 7beta-HSDH sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The 7beta-HSDH gene is then cloned from the plasmid by PCR amplification. The restriction site BamH I is added to the upstream primer, and Hind III is added to the downstream primer.
The CBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The CBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind III added to the upstream primer, and Xhol I added to the downstream primer.
Firstly, the 7beta-HSDH gene is inserted into the modified pET-28x at BamH I and Hind III, and CBD at Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac operator--RBS--His-tag--7beta-HSDH--CBD--T7 terminator, is retrieved from this plasmid by PCR amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I and Pst I.
[[file:Psb1c3-cbd-7b.jpeg|300px]]
[Fig. 1. pSB1C3--CBD--7beta-HSDH]
Usage and Biology
7beta-HSDH(7beta-hydroxysteroid dehydrogenase) catalyzes the reduction of hydroxysteroids at C-7 position and back, as it converts NADPH to NDAP+ and back. It catalyzes the reduction of the intermediate product 7-oxo-LAC (7-ketolithocholic acid) into UDCA, the final product, where the carbonyl at C-7 position becomes hydroxyl.[1]
CBD (cellulose binding domain) is able to bind to cellulose. When connected to 7beta-HSDH, CBD is able to immobilize the enzyme 7beta-HSDH after expression, by binding to the gauze inside the solution on its cellulose.[2]
The function of cellulose binding domain
The function of CBD is tested by connecting CBD gene with GFP gene in pET28x. The GFP-CBD fusion protein is expressed and mixed with a gauze piece. The green fluorescent on the gauze is not significantly reduced after washing, proving that the CDB is well functioned. In comparison, no green fluorescent is left after washing the gauze mixed with GFP-ChBD (Chintin binding domain).
[[file:cbd-before.jpeg|150px]]
[Fig. 2. GFP-CBD on gauze before washing]
[[file:cbd-after.jpeg|150px]]
[Fig. 3. GFP-CBD on gauze after washing]
[[file:chbd-before.jpeg|150px]]
[Fig. 4. GFP-ChBD on gauze before washing]
[[file:chbd-after.jpeg|150px]]
[Fig. 5. GFP-ChBD on gauze after washing]
Expression and Immobilization[2]
The constructed pET28x--7beta-HSDH--CBD plasmid is transformed into BL21(DE3) E.coli for expression. After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is broken by ultrasonication.
The resulted bacteria solution is diluted to a certain concentration and mixed with gauze piece, and the gauze piece is washed three times by ddH2O afterwards. As a result, the CBD protein binds to the cellulose on gauze, and the enzyme is successfully immobilized.
Enzyme Activity
The reduction of 7OXO-LCA to UDCA
[[file:reaction1-2.jpeg|400px]]
[Fig. 6. Reaction process]
7β-HSDH-CBD is an NADPH dependent enzyme from Ruminococcus Torques. The 3mL reaction contains 150mM phosphate buffer(pH 8.0), 10mM UDCA, 0.2mM NADPH. The reaction started when the solution is combined with 7β-HSDH-CBD-enzyme-binding gauze that in different concentration, includes 80ul, 120ul, 160ul liquid supernatant of ultrasonication bacteria solution. The control group was testify under the same solution and condition but using pET28x-CBD liquid supernatant of ultrasonication bacteria solution to bind with gauze in the concentration of 160ul. Before adding the gauze into the solution, the gauze was washed by ddH2O for 3 times in order to purify the enzyme.
The 7-OXO-LCA was convert to UDCA by Taking a a pair of hydrogen(2H+and 2e-)from the NADPH and form 7-hydroxyl group (B position). The co-enzyme NADPH is the donor of the a pair of hydrogen(2H+and 2e-), and was transformed into NADP+. However, to make the reaction easy to testify, the reverse reaction that transforming UDCA to 7OXO-LCA was employed to prove the function of 7B in which The B position 7-hydroxyl group loose a pair of hydrogen(2H+and 2e-) and from the 7-carbonyl group, and NADP+ is the acceptor of the a pair of hydrogen(2H+and 2e-), to form NADPH The the synthesized NADHP can be determined spectrophotometrically at340 nm (ε = 6.22 mM-1 cm-1) and room temperature. One unit of activity is defined as the amount of enzyme catalyzing the synthesize of 1 mmol of NADPH per min under the assay conditions used.
Result
[[file:result1-3.jpeg|400px]]
[Fig. 7. Result]
Reduction of 7-oxo-LCA to UDCA using 7β-HSDH and GDH(NADPH regeneration)
[[file:reaction2-2.jpeg|400px]]
[Fig. 8. Reaction process]
The 3mL reaction solution containing 150 mM phosphate buffer(pH 8.0), 10 mM UDCA, 30 mM glucose, 0.2mM NADP+, combined with 1U/ml 7β-HSDH and 5U/ml GDH at room temperature.
The bioconversion experiment was monitored via HPLC measurements. The sample was analyzed by UV detection at 210nm. We testify the synthesize of 7-oxo-LCA and the decrease of UDCA, using a mobile phase of methanol–water mixture (final ratio 80:20,pH 3.5 with phosphoric acid) using chromatographic column C18 .
Result
[[file:hplc2-1.jpeg|500px]]
[Fig. 9. Absorbance of NADPH]
[[file:hplc2-2.jpeg|400px]]
[Fig. 10. Peak area of 7oxo-LCA sample in different concentration]
[[file:hplc2-3.jpeg|500px]]
[Fig. 11. Time course]
According to the HPLC result after 90minute, there is no significant increase of 7oxo-LCA, as a result, most of the UDCA has been transformed into 7oxo-LCA. And according to the HPLC the final yielding rate is 93%.
According to the Absorbance of NADPH that shown in figure, the absorbance is decreased significantly after 90 minute dual to the depleted UDCA that stop the conversion of UDCA to 7oxo-LCA and NADP+ synthesize. Because of the abundant amount of glucose in the solution, the GDH that works on glucose still regenerate the NADPH by taking a pair of hydrogen from glucose until most of the NADP+ transformed into NADPH.
Reference
[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process Biochemistry, Elsevier, 2015.
[2] Etai Shpigel, Arie Goldlust, Gilat Efroni, Amos Avraham, Adi Eshel, Mara Dekel, Oded Shoseyov: Immobilization of Recombinant Heparinase I Fused to Cellulose-Binding Domain, 1999.
--CBD-LDH
CBD-LDH (T7 promoter--lac operator--RBS--His-tag--LDH--CBD--T7 terminator)
This device codes for the LDH-CBD fusion protein.
Construct
The vector of LDH-CBD for its expression is pET-28x. It is formed by modifying the restriction enzyme sites EcoR I and Xba I of vector pET-28a.
The LDH sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The LDH gene is then cloned from the plasmid by PCR amplification. The restriction site BamH I is added to the upstream primer, and Hind III is added to the downstream primer.
The CBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The CBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind III added to the upstream primer, and Xhol I added to the downstream primer.
Firstly, the LDH gene is inserted into the modified pET-28x at BamH I and Hind III, and CBD at Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac operator--RBS--His-tag--LDH--CBD--T7 terminator, is retrieved from this plasmid by PCR amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I and Pst I.
[[file:Psb1c3-cbd-ldh.jpeg|300px]]
[Fig. 1. pSB1C3-CBD-LDH]
Usage and Biology
LDH(lactate dehydrogenase) catalyzes the conversion of lactate to pyruvic acid and back, as it converts NAD+ to NADH and back. It is used to catalyze the reduction of pyruvate into lactate, with the presence of NADH, which is oxidized into NAD+ at the same time.[1]
CBD (cellulose binding domain) is able to bind to cellulose. When connected to LDH, CBD is able to immobilize the enzyme LDH after expression, by binding to the gauze inside the solution on its cellulose.[2]
The function of cellulose binding domain
The function of CBD is tested by connecting CBD gene with GFP gene in pET28x. The GFP-CBD fusion protein is expressed and mixed with a gauze piece. The green fluorescent on the gauze is not significantly reduced after washing, proving that the CDB is well functioned. In comparison, no green fluorescent is left after washing the gauze mixed with GFP-ChBD (Chintin binding domain).
[[file:cbd-before.jpeg|150px]]
[Fig. 2. GFP-CBD on gauze before washing]
[[file:cbd-after.jpeg|150px]]
[Fig. 3. GFP-CBD on gauze after washing]
[[file:chbd-before.jpeg|150px]]
[Fig. 4. GFP-ChBD on gauze before washing]
[[file:chbd-after.jpeg|150px]]
[Fig. 5. GFP-ChBD on gauze after washing]
Expression and Immobilization[2]
The constructed pET28x-LDH plasmid is transformed into BL21(DE3) E.coli for expression. After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is broken by ultrasonication.
The resulted bacteria solution is diluted to a certain concentration and mixed with gauze piece and the gauze piece is washed three times by ddH2O afterwards. As a result, the CBD protein binds to the cellulose on gauze, and the enzyme is successfully immobilized.
Enzyme Activity
The NAD+ regeneration
LDH is an NADH dependent enzyme from Lactobacillus delbrueckii subspecies bulgaricus. The 3mL reaction consists of 150mM phosphate buffer(pH 8.0), 25mM Lactose, 0.2mM NAD+. The reaction started when the solution is combined with LDH-CBD-enzyme-binding gauze that in different concentration, includes 5ul, 10ul, and 15ul liquid supernatant of ultrasonication bacteria solution. The control group was testify under the same solution and condition but using pET28x-CBD liquid supernatant of ultrasonication bacteria solution to bind with gauze in the concentration of 15ull. Before adding the gauze into the solution, the gauze was washed by ddH2O for 3 times in order to purify the enzyme.
The LDH works on pyruvate and take a pair of hydrogen(2H+and 2e-) from NADH to form lactate. The NAD+ was regenerate through this reaction. Since the NADH can be testified under 340nm of ultraviolet light, the reverse reaction of LDH is employed to testify its function. The enzyme activity was determined spectrophotometrically atv340 nm (ε = 6.22 mM-1 cm-1) and room temperature by measuring the synthesize of NADH. One unit of activity is defined as the amount of enzyme catalyzing the synthesize of 1 mmol of NADH per min under the assay conditions used.
Result
[[file:result1-2.jpeg|400px]]
[Fig. 6. Result]
Oxidation of CDCA to 7-oxo-LCA using E. coli 7alpha-HSDH and NAD+ regeneration
[[file:reaction2-1.jpeg|400px]]
[Fig. 7. Reaction process]
CDCA was converted in a 3mL solution containing 150 mM phosphate buffer(pH 8.0), 10 mM CDCA, 30 mM sodium pyruvate, 0.25mM NAD+, combined with 3U/ml LDH and1 1 U/ml E. coli DH5a 7a-HSDH-CBD at room temperature.
The bioconversion experiments were monitored via HPLC measurements. The sample was analyzed by UV detection at 210 nm, using a mobile phase of methanol–water mixture (final ratio 80:20,pH 3.5 with phosphoric acid) using chromatographic column C18.
Result
As shown in figure, the transformation was complete after 2.5h.
[[file:hplc1-1.jpeg|500px]]
[Fig. 9. Absorbance of NADH]
[[file:hplc1-2.jpeg|400px]]
[Fig. 10. Peak area of 7oxo-LCA sample in different concentration]
[[file:hplc1-3.jpeg|500px]]
[Fig. 11. Time course]
According to the HPLC after 150minute, there is no significant increase of 7oxo-LCA, as a result, most of the CDCA has been transformed into 7oxo-LCA. And according to the HPLC the final yielding rate is 94%.
According to the Absorbance of NADH that shown in figure, the absorbance is decreased significantly after 150 minute dual to the depleted CDCA that stop the conversion of CDCA to 7oxo-LCA and NADH synthesize. Because of the abundant amount of pyruvate in the solution, the LDH that works on pyruvate still regenerate the NAD+ by taking a pair of hydrogen from NADH until most of the NADH synthesized transformed into NAD+.
Reference
[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process Biochemistry, Elsevier, 2015.
[2] Etai Shpigel, Arie Goldlust, Gilat Efroni, Amos Avraham, Adi Eshel, Mara Dekel, Oded Shoseyov: Immobilization of Recombinant Heparinase I Fused to Cellulose-Binding Domain, 1999.
--CBD-GDH
CBD-GDH (T7 promoter--lac operator--RBS--His-tag--GDH--CBD--T7 terminator)
This device codes for the GDH-CBD fusion protein.
Construct
The vector of GDH-CBD for its expression is pET-28x. It is formed by modifying the restriction enzyme sites EcoR I and Xba I of vector pET-28a.
The GDH sequence is cloned from the genome of Bacillus subtilis through PCR amplification, using the primers designed and synthesized based on its sequence. The restriction site BamH I is added to the upstream primer, and Hind III is added to the downstream primer.
The CBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The CBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind III added to the upstream primer, and Xhol I added to the downstream primer.
Firstly, the GDH gene is inserted into the modified pET-28x at BamH I and Hind III, and CBD at Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac operator--RBS--His-tag--GDH--CBD--T7 terminator, is retrieved from this plasmid by PCR amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I and Pst I.
[[file:Psb1c3-cbd-gdh.jpeg|300px]]
[Fig. 1. pSB1C3-CBD-GDH]
Usage and Biology
GDH(glucose dehydrogenase) catalyzes the conversion of beta-D-glucose to D-glucono-1,5-lactone and back, as it converts NADP+ to NADPH and back. It is used to catalyze the oxidation of beta-D-glucose into D-glucono-1,5-lactone and reduce NADP+ into NADPH during the process.[1]
CBD (cellulose binding domain) is able to bind to cellulose. When connected to GDH, CBD is able to immobilize the enzyme GDH after expression, by binding to the gauze inside the solution on its cellulose.[2]
The function of cellulose binding domain
The function of CBD is tested by connecting CBD gene with GFP gene in pET28x. The GFP-CBD fusion protein is expressed and mixed with a gauze piece. The green fluorescent on the gauze is not significantly reduced after washing, proving that the CDB is well functioned. In comparison, no green fluorescent is left after washing the gauze mixed with GFP-ChBD (Chintin binding domain).
[[file:cbd-before.jpeg|150px]]
[Fig. 2. GFP-CBD on gauze before washing]
[[file:cbd-after.jpeg|150px]]
[Fig. 3. GFP-CBD on gauze after washing]
[[file:chbd-before.jpeg|150px]]
[Fig. 4. GFP-ChBD on gauze before washing]
[[file:chbd-after.jpeg|150px]]
[Fig. 5. GFP-ChBD on gauze after washing]
Expression and Immobilization[2]
The constructed pET28x-GDH plasmid is transformed into BL21(DE3) E.coli for expression. After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is broken by ultrasonication.
The resulted bacteria solution is diluted to a certain concentration and mixed with gauze piece and the gauze piece is washed three times by ddH2O afterwards. As a result, the CBD protein binds to the cellulose on gauze, and the enzyme is successfully immobilized.
Enzyme Activity
Regeneration of NADPH
GDH is an NADPH dependent enzyme from bacillus subtilis. The 3mL reaction consists of 150mM phosphate buffer(pH 6.5), 30mM glucose, 0.2mM NADP+. The reaction started when the solution is combined with GDH-CBD-enzyme-binding gauze that in different concentration, includes 80ul, 120ul, and 160ul liquid supernatant of ultrasonication bacteria solution. The control group was testify under the same solution and condition but using pET28x-CBD liquid supernatant of ultrasonication bacteria solution to bind with gauze in the concentration of 160ul. Before adding the gauze into the solution, the gauze was washed by ddH2O for 3 times in order to purify the enzyme.
The GDH works on glucose and take a pair of hydrogen(2H+and 2e-) from glucose and add to NADP+. The NADPH was regenerate through this reaction. Since the NADPH can be testified under 340nm of ultraviolet light, the enzyme activity was determined spectrophotometrically atv340 nm (ε = 6.22 mM-1 cm-1) and room temperature by measuring the synthesize of N. One unit of activity is defined as the amount of enzyme catalyzing the synthesize of 1 mmol of NADH per min under the assay conditions used.
Result
[[file:result1-4.jpeg|400px]]
[Fig. 6. Result]
Reduction of 7-oxo-LCA to UDCA using 7β-HSDH and GDH(NADPH regeneration)
[[file:reaction2-2.jpeg|400px]]
[Fig. 7. Reaction process]
The 3mL reaction solution containing 150 mM phosphate buffer(pH 8.0), 10 mM UDCA, 30 mM glucose, 0.2mM NADP+, combined with 1U/ml 7β-HSDH and 5U/ml GDH at room temperature.
The bioconversion experiment was monitored via HPLC measurements. The sample was analyzed by UV detection at 210nm. We testify the synthesize of 7-oxo-LCA and the decrease of UDCA, using a mobile phase of methanol–water mixture (final ratio 80:20,pH 3.5 with phosphoric acid) using chromatographic column C18 .
Result
[[file:hplc2-1.jpeg|500px]]
[Fig. 9. Absorbance of NADPH]
[[file:hplc2-2.jpeg|400px]]
[Fig. 10. Peak area of 7oxo-LCA sample in different concentration]
[[file:hplc2-3.jpeg|500px]]
[Fig. 11. Time course]
According to the HPLC result after 90minute, there is no significant increase of 7oxo-LCA, as a result, most of the UDCA has been transformed into 7oxo-LCA. And according to the HPLC the final yielding rate is 93%.
According to the Absorbance of NADPH that shown in figure, the absorbance is decreased significantly after 90 minute dual to the depleted UDCA that stop the conversion of UDCA to 7oxo-LCA and NADP+ synthesize. Because of the abundant amount of glucose in the solution, the GDH that works on glucose still regenerate the NADPH by taking a pair of hydrogen from glucose until most of the NADP+ transformed into NADPH.
Reference
[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process Biochemistry, Elsevier, 2015.
[2] Etai Shpigel, Arie Goldlust, Gilat Efroni, Amos Avraham, Adi Eshel, Mara Dekel, Oded Shoseyov: Immobilization of Recombinant Heparinase I Fused to Cellulose-Binding Domain, 1999.
--CBD-GFP
CBD-GFP (T7 promoter--lac operator--RBS--His-tag--GFP--CBD--T7 terminator)
This device codes for the GFP-CBD fusion protein.
Construct
The vector of GFP-CBD for its expression is pET-28x. It is formed by modifying the restriction enzyme sites EcoR I and Xba I of vector pET-28a.
The GFP sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The GFP gene is then cloned from the plasmid by PCR amplification. The restriction site BamH I is added to the upstream primer, and Hind III is added to the downstream primer.
The CBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The CBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind III added to the upstream primer, and Xhol I added to the downstream primer.
Firstly, the GFP gene is inserted into the modified pET-28x at BamH I and Hind III, and CBD at Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac operator--RBS--His-tag--GFP--CBD--T7 terminator, is retrieved from this plasmid by PCR amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I and Pst I.
[[file:Psb1c3-cbd-gfp.jpeg|300px]]
[Fig. 1. pSB1C3-CBD-GFP]
Usage and Biology
This part is used to test the function of CBD (cellulose binding domain), which is able to bind to cellulose.[1]
Expression
The constructed pET28x-GFP-CBD plasmid is transformed into BL21(DE3) E.coli for expression. After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is broken by ultrasonication.
Proof of the CBD function
The resulted solution after expression is mixed with a gauze piece, and the green color on gauze is recorded. Then the gauze is washed three times using ddH2O, but the green fluorescent on it is not significantly reduced, proving that the CDB is well functioned. In comparison, no green fluorescent is left after washing the gauze mixed with GFP-ChBD (Chintin binding domain).
[[file:cbd-before.jpeg|150px]]
[Fig. 2. GFP-CBD on gauze before washing]
[[file:cbd-after.jpeg|150px]]
[Fig. 3. GFP-CBD on gauze after washing]
[[file:chbd-before.jpeg|150px]]
[Fig. 4. GFP-ChBD on gauze before washing]
[[file:chbd-after.jpeg|150px]]
[Fig. 5. GFP-ChBD on gauze after washing]
Reference
[1] Etai Shpigel, Arie Goldlust, Gilat Efroni, Amos Avraham, Adi Eshel, Mara Dekel, Oded Shoseyov: Immobilization of Recombinant Heparinase I Fused to Cellulose-Binding Domain, 1999.
contribution
We improved the description of ChBD and CBD on previous part pages.
-ChBD (Part:BBa_T2028)
Proof of the ChBD function
The resulted solution after expression is mixed with chitin powder. Also, the solution with GFP-ChBD but without chitin, solution with the presence of GFP and chitin, and water in comparison, are presented.
Tube 3 contains GFP-ChBD, with a significant green color in the solution. After adding chitin powder inside the tube, the color in the solution disappears and shows on the chitin powder, as presented in tube 2. It proves that the ChBD successfully binds the GFP onto the chitin powder. In contrast, the color of the solution is not changed when adding chitin powder into the GFP solution without ChBD. As a result, the ChBD is well functioned.
[[file:chbd.jpeg|250px]]
[Fig.2. ChBD function: tube1, GFP and chitin; tube2, GFP-ChBD and chitin; tube3, GFP-ChBD; tube4, water]
-CBD (Part:BBa_K1979001)
Proof of the CBD function
The resulted solution after expression is mixed with a gauze piece, and the green color on gauze is recorded. Then the gauze is washed three times using ddH2O, but the green fluorescent on it is not significantly reduced, proving that the CDB is well functioned. In comparison, no green fluorescent is left after washing the gauze mixed with GFP-ChBD (Chintin binding domain).
[[file:cbd-before.jpeg|150px]]
[Fig. 2. GFP-CBD on gauze before washing]
[[file:cbd-after.jpeg|150px]]
[Fig. 3. GFP-CBD on gauze after washing]
[[file:chbd-before.jpeg|150px]]
[Fig. 4. GFP-ChBD on gauze before washing]
[[file:chbd-after.jpeg|150px]]
[Fig. 5. GFP-ChBD on gauze after washing]
