title: Premethylation of foreign DNA improves integrative transformation efficiency in Synechocystis sp. strain PCC 6803.
abstract: Restriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium, Synechocystis sp. strain PCC 6803 (Synechocystis 6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e., sll0729 (gene M) and slr0214 (gene C), were cloned from the chromosome of Synechocystis 6803 and expressed in Escherichia coli harboring an integration plasmid. After premethylation treatment in E. coli, the integration plasmid was extracted and used for transformation of Synechocystis 6803. The results showed that although expression of methyltransferase M had little impact on the transformation of Synechocystis 6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation of Synechocystis 6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5' untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency in Synechocystis 6803. Since premethylating foreign DNA prior to transforming Synechocystis avoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation of Synechocystis 6803.
authors.affiliation: Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, Arizona, USA Biological Design Graduate Program, Arizona State University, Tempe, Arizona, USA National Renewable Energy Laboratory, Golden, Colorado, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Wang
authors.affiliation: National Renewable Energy Laboratory, Golden, Colorado, USA.
authors.forename: Jianping
authors.initials: J
authors.lastname: Yu
authors.affiliation: Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, Arizona, USA wwzhang8@tju.edu.cn deirdre.meldrum@asu.edu.
authors.forename: Weiwen
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, Arizona, USA wwzhang8@tju.edu.cn deirdre.meldrum@asu.edu.
authors.forename: Deirdre R
authors.initials: DR
authors.lastname: Meldrum
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D003001:Cloning, Molecular; D019175:DNA Methylation / Q000235:genetics*; D015254:DNA Modification Methylases / Q000096:biosynthesis / Q000235:genetics; D061269:DNA Transformation Competence / Q000235:genetics*; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D010957:Plasmids / Q000235:genetics; D046939:Synechocystis / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial / Q000235:genetics*
chemical_list: D020121:5' Untranslated Regions; D004269:DNA, Bacterial; D015254:DNA Modification Methylases