Synthetic Biology Journal ›› 2023, Vol. 4 ›› Issue (4): 840-851.DOI: 10.12211/2096-8280.2022-048
• Research Article • Previous Articles
Wanqiu LIU, Xiangyang JI, Huiling XU, Yicong LU, Jian LI
Received:
2022-09-06
Revised:
2022-12-01
Online:
2023-09-14
Published:
2023-08-31
Contact:
Jian LI
刘晚秋, 季向阳, 许慧玲, 卢屹聪, 李健
通讯作者:
李健
作者简介:
基金资助:
CLC Number:
Wanqiu LIU, Xiangyang JI, Huiling XU, Yicong LU, Jian LI. Cell-free protein synthesis system enables rapid and efficient biosynthesis of restriction endonucleases[J]. Synthetic Biology Journal, 2023, 4(4): 840-851.
刘晚秋, 季向阳, 许慧玲, 卢屹聪, 李健. 限制性内切酶的无细胞快速制备研究[J]. 合成生物学, 2023, 4(4): 840-851.
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URL: https://synbioj.cip.com.cn/EN/10.12211/2096-8280.2022-048
引物名称 | 序列 | 用途 |
---|---|---|
linearPro_F | CCTACAGCGTGAGCATTG | 扩增启动子片段 |
linearPro_R | CATATGGTGATGATGATG | |
linearTer_F | GTCGACCGGCTGCTAACA | 扩增终止子片段 |
linearTer_R | CGGATTCAGTCGTCACTCA | |
SUMOPro_F | GGATCTCGACGCTCTCCCT | 扩增启动子+SUMO标签片段 |
SUMOPro_R | AGGTCCCTGAAACAGGACCTCTAAACCACCAATCTGTTCTCTG | |
SUMO_mut_F | TACGACGGTATTCGTATTCAAGCTGATCAGAC | 去除pSUMO质粒中的EcoRⅠ酶切位点 |
SUMO_mut_R | CAGCTTGAATACGAATACCGTCGTACAAGAATC | |
EcoRⅠ_SUMO_F | TTAGAGGTCCTGTTTCAGGGACCTAGCAACAAAAAACAGAGC | 扩增EcoRⅠ基因片段 |
EcoRⅠ_SUMO_R | CATCATCATCACCATATGAGCAACAAAAAACAGAGC | |
sfGFP_SUMO_F | ATTGGTGGTACCGAGCTCATGAGCAAAGGTGAAGAA | 构建质粒pSUMO-sfGFP |
sfGFP_SUMO_R | GAGTGCGGCCGCAAGCTTTTATTTTTCGAACTGCGG | |
BamHⅠ_F | CATCATCATCACCATATGAAAGTGGAAAAAGA | 扩增BamHⅠ基因片段 |
BamHⅠ_R | TGTTAGCAGCCGGTCGACTTATTTGTTTTCCACTTTATC | |
BsaⅠ_F | TTAGAGGTCCTGTTTCAGGGACCTATGGCAAAAAAGCGGAA | 扩增BsaⅠ基因片段 |
BsaⅠ_R | TGTTAGCAGCCGGTCGACTTAATCCAGATCCGCAAA |
Table 1 Oligonucleotide primers used in this study
引物名称 | 序列 | 用途 |
---|---|---|
linearPro_F | CCTACAGCGTGAGCATTG | 扩增启动子片段 |
linearPro_R | CATATGGTGATGATGATG | |
linearTer_F | GTCGACCGGCTGCTAACA | 扩增终止子片段 |
linearTer_R | CGGATTCAGTCGTCACTCA | |
SUMOPro_F | GGATCTCGACGCTCTCCCT | 扩增启动子+SUMO标签片段 |
SUMOPro_R | AGGTCCCTGAAACAGGACCTCTAAACCACCAATCTGTTCTCTG | |
SUMO_mut_F | TACGACGGTATTCGTATTCAAGCTGATCAGAC | 去除pSUMO质粒中的EcoRⅠ酶切位点 |
SUMO_mut_R | CAGCTTGAATACGAATACCGTCGTACAAGAATC | |
EcoRⅠ_SUMO_F | TTAGAGGTCCTGTTTCAGGGACCTAGCAACAAAAAACAGAGC | 扩增EcoRⅠ基因片段 |
EcoRⅠ_SUMO_R | CATCATCATCACCATATGAGCAACAAAAAACAGAGC | |
sfGFP_SUMO_F | ATTGGTGGTACCGAGCTCATGAGCAAAGGTGAAGAA | 构建质粒pSUMO-sfGFP |
sfGFP_SUMO_R | GAGTGCGGCCGCAAGCTTTTATTTTTCGAACTGCGG | |
BamHⅠ_F | CATCATCATCACCATATGAAAGTGGAAAAAGA | 扩增BamHⅠ基因片段 |
BamHⅠ_R | TGTTAGCAGCCGGTCGACTTATTTGTTTTCCACTTTATC | |
BsaⅠ_F | TTAGAGGTCCTGTTTCAGGGACCTATGGCAAAAAAGCGGAA | 扩增BsaⅠ基因片段 |
BsaⅠ_R | TGTTAGCAGCCGGTCGACTTAATCCAGATCCGCAAA |
Fig. 2 Cell-free production of restriction endonuclease EcoRⅠ(Western blot and SDS-PAGE analyses of cell-free expressed and purified restriction endonuclease EcoRⅠ) CE—cell extract of BL21 Star (DE3); CE+ —cell extract of BL21 Star (DE3) with chaperone (pG-KJE8); T—total protein; S—soluble protein; M—marker; FT—flow throughout sample; W—washed sample; Eluate—eluted sample
Fig. 5 Cell-free production of restriction endonucleases BamHⅠ and BsaⅠ(Western Blot and SDS-PAGE analyses of cell-free expressed and purified restriction endonucleases BamHⅠ and BsaⅠ)
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