The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.
T7 RNA聚合酶T7启动子鱼腥藻Anabaena sp. PCC 7120人粒细胞集落刺激因子电击转化三亲结合T7 RNA polymeraseT7 promoterAnabaena sp. PCC 7120hG-CSFelectroporationtriparental conjugative transfer北京中医药大学发展基金2020071420021国家自然科学基金81774014北京中医药大学发展基金(No. 2020071420021),国家自然科学基金(No. 81774014)资助Beijing University of Chinese Medicine2020071420021National Natural Science Foundation of China81774014Development Fund of Beijing University of Chinese Medicine (No. 2020071420021), National Natural Science Foundation of China (No. 81774014)
采用三亲接合转移法[5]。(1) E. coli的准备:在含有相应抗生素的LB培养液中,分别接种含穿梭表达载体pRL-T7-hG-CSF和辅助质粒的HB101菌,37 ℃过夜培养,离心收集沉淀,等体积混合后重悬于LB培养液中。(2)蓝藻的准备:三亲接合转移前更换新鲜培养液,离心收集野生藻与含T7 RNA聚合酶转基因藻,洗涤后重悬于BG11培养液中,并作梯度稀释。(3)接合转移:将藻细胞与细菌以适当的比例混合均匀,涂布于纤维酯微孔滤膜上,先在不含抗生素的培养平板上培养,然后连膜转移至含卡那霉素或卡那霉素和氯霉素的培养基上继续照光培养,每周更换一次平板,并逐步提高抗生素浓度,直至转基因藻平板出现阳性克隆而野生藻死亡。
The acquisition of Tac-T7 RNAP-CmR gene sequences. (A) The acquisition of Tac promoter gene sequence. M: 100 bp DNA ladder; 1: PCR product of Tac promoter. (B) The acquisition of T7 RNA polymerase with 6×His tag gene sequence. M: 1 kb DNA ladder; 1: PCR product of T7 RNA polymerase; 2: PCR product of T7 RNA polymerase with 6×His tag. (C) The acquisition of chloramphenicol resistant gene sequence. M: DNA marker Ⅳ; 1: PCR product of chloramphenicol resistant gene. (D) The acquisition of Tac-T7 RNAP-CmR gene sequence. M: 1 kb DNA ladder; 1: gene of Tac-T7 RNAP-CmR by overlap PCR.
The construction and identification of the site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2. (A) The acquisition of homologous arms. M: 1 kb DNA ladder; 1: PCR product of homologous arm F1; 2: PCR product of homologous arm F2. (B) The acquisition of homologous arm F1 with restriction enzyme site. M: 1 kb DNA ladder; 1: pMD19-T-F1 digested by Hind Ⅲ and SacⅠ. (C) The acquisition of homologous arm F2 with restriction enzyme site. M: 1 kb DNA ladder; 1: pEASY-T3-F2 digested by NotⅠ. (D) The acquisition of the linearized plasmid pEASY-T1- Tac-T7RNAP-CmR. M: 1 kb DNA ladder; 1: pEASY-T1-Tac-T7RNAP-CmR digested by Hind Ⅲ andSacⅠ. (E) The acquisition of the site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2. M: 1 kb DNA ladder; 1: The recombinant plasmid pEASY-T1-F1-TacT7RNAPCmR-F2. (F) The identification of the site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2. M: 1 kb DNA ladder; 1: pEASY-T1-F1-TacT7RNAPCmR-F2 digested by SacⅠ and ApaⅠ; 2: pEASY-T1-F1-TacT7RNAPCmR-F2 digested by Hind Ⅲ and ApaⅠ.
The construction and identification of the shuttle expression vector pRL-T7-hG-CSF. (A) The acquisition of T7-hG-CSF gene fragment with restriction enzyme site. M: 1 kb DNA ladder; 1: T5-T7-hG-CSF; 2: T5-T7- hG-CSF digested by EcoRⅠand BamHⅠ. (B) The acquisition of the linearized plasmid pRL489. M: 1 kb DNA ladder; 1: pRL489 digested by EcoRⅠ and BamHⅠ; 2: pRL489. (C) The acquisition and identification of the shuttle expression vector pRL-T7-hG-CSF. m: 100 bp DNA ladder; M: 1 kb DNA ladder; 1: the recombinant plasmid pRL-T7-hG-CSF; 2: pRL-T7-hG-CSF digested by EcoRⅠand BamHⅠ.
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