材料与试剂

1. 0.22 μm无菌针孔过滤器 (Sangon Biotech, catalog number: F513134-0001)
2. 高保真DNA聚合酶 (2x Phanta Max Master Mix, Vazyme, catalog number: P515-02)
3. DNA聚合酶 (Taq Master Mix (Dye Plus), Vazyme, catalog number: P112-02)
4. DNA Marker (Fermentas, GeneRulerTM 100 by Plus DNA Ladder)
5. 蛋白Marker (Fermentas, PageRulerTM Prestained Protein Ladder)
6. T4 DNA连接酶 (TransGen Biotech, catalog number: FL101-01)
7. 限制性内切酶 EcoRI (TaKaRa, catalog number: 1040BH)；HindIII (TaKaRa, catalog number: 1060B)
8. 质粒提取试剂盒 (Omega, Plasmid Mini Kit I (200) 200T)
9. PCR产物纯化试剂盒 (Corning, Axygen, catalog number: AP-PCR-50)
10. 割胶回收试剂盒 (Corning, Axygen, catalog number: AP-GX-50)
11. 反转录试剂盒 (Toyobo, ReverTra Ace Kit)
12. 酵母膏
13. 胰蛋白胨
14. 氨苄青霉素
15. 卡那霉素
16. IPTG (BBI)
17. Ni-NTA agarose (Sangon Biotech, catalog number: C600033)
18. 40%丙烯酰胺/甲叉双丙烯酰胺 (29:1) 溶液 (Sangon Biotech, catalog number: B546012-0500)
19. EDTA·2H₂O (Sangon Biotech, catalog number: A500838-0500)
20. Tris (Sangon Biotech, catalog number: A610195-0500)
21. 冰醋酸 (国药, catalog number: 64-19-7)
22. 3,5-二硝基水杨酸 (3,5-Dinitrosalicylic acid, DNS) (Sangon Biotech, catalog number: A500420-0100)
23. NaOH (国药, catalog number: 10019718)
24. 酒石酸钾钠 (Sangon Biotech, catalog number: A500881-0500)
25. NaCl (Sangon Biotech, catalog number: A501218-0001)
26. KCl (Sangon Biotech, catalog number: A100395-0500)
27. Na₂HPO₄ (Sangon Biotech, catalog number: A501727-0500)
28. KH₂PO₄ (Sangon Biotech, catalog number: A100781-0500)
29. SDS (Sangon Biotech, catalog number: A500228-0001)
30. 甘氨酸 (Sangon Biotech, catalog number: A610235-0005)
31. 过硫酸铵 (Sangon Biotech, catalog number: A500857-0100)
32. 溴酚蓝 (Sangon Biotech, catalog number: A500922-0025)
33. 甘油 (Sangon Biotech, catalog number: A100854-0500)
34. β-巯基乙醇 (Macklin, catalog number: M6230-25ml)
35. 甲醇 (国药, catalog number: 10014118)
36. 考马斯亮蓝 R-250 (Sangon Biotech, catalog number: A100472-0025)
37. LB (Luria-Bertani) (见溶液配方)
38. 0.5 mol/L EDTA (见溶液配方)
39. 50x TAE母液 (见溶液配方)
40. 100 mg/ml Kan (见溶液配方)
41. IPTG储存液 (见溶液配方)
42. 3,5-二硝基水杨酸溶液 (见溶液配方)
43. PBS缓冲液 (pH 7.4) (见溶液配方)
44. 1.5 mol/L Tris-HCl (pH 8.8) (见溶液配方)
45. 1 mol/L Tris-HCl (pH 6.8) (见溶液配方)
46. 10% SDS (w/v) (见溶液配方)
47. 50%甘油 (v/v) (见溶液配方)
48. 5x SDS-PAGE缓冲液 (见溶液配方)
49. 10%过硫酸铵AP (w/v) (见溶液配方)
50. 1%溴酚蓝 (w/v) (见溶液配方)
51. 5x SDS上样缓冲液 (见溶液配方)
52. R250染色液 (见溶液配方)
53. SDS-PAGE脱色液 (见溶液配方)
54. NaH₂PO₄母液 (500 mmol/L) (见溶液配方)
55. NaCl母液 (3 mol/L) (见溶液配方)
56. 1 mol/L咪唑 (见溶液配方)
57. Washing buffer 1 (含20 mmol/L咪唑) (见溶液配方)
58. Washing buffer 2 (含50 mmol/L咪唑) (见溶液配方)
59. Eluent buffer (含250 mmol/L咪唑) (见溶液配方)
60. 转膜缓冲液 (见溶液配方)
61. TBST (见溶液配方)
62. 封闭液 (见溶液配方)
    

仪器设备

1. 离心机 (Eppendorf, model: Centrifuge MiniSpin)
2. PCR仪 (Eppendorf, model: Mastercycler Pros)
3. 电子分析天平 (梅特勒-托利多, model: EL204)
4. 微波炉 (格兰仕, model: D80D23NTP-7T)
5. 空气恒温摇床 (宁波科技园区新江南仪器有限公司, model: KYC-100B)
6. 生化培养箱 (宁波东南仪器有限公司, model: LRH-50)
7. 洁净工作台 (苏州市金净净化设备科技有限公司, model: JJ-CJ-ETD)
8. 水平脱色摇床 (苏州威尔实验用品有限公司, model: ZD-9560)
9. 凝胶成像系统 (Molecular imager, model: GeL.Dcode XR+)
10. 立式压力蒸汽灭菌器 (上海博讯实业有限公司医疗设备厂, model: YXQ-75SII)
11. 超声波破碎仪 (浙江宁波/新芝, model: JY98-IIIDN)
    

软件和数据库

1. dbCAN HMMdb v9 (http://bcb.unl.edu/dbCAN2/index.php)
2. Carbohydrate-Active enZYmes Database (http://www.cazy.org)
3. Pfam Database (http://pfam.xfam.org/search#tabview=tab0)
4. Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/)
5. Oligo 6.0

实验步骤