Construction of a broad host range expression plasmid vector by Golden Gate cloning
- Author:
Yuh Leng Teo
1
;
Wai Keat Toh
1
;
Xin Yen Tor
1
;
Chai-Ling Ho
2
;
Pek Chin Loh
1
;
Hann Ling Wong
1
Author Information
- Publication Type:Other Types
- Keywords: Plasmid; Expression vector; Broad host range; Golden Gate cloning
- MeSH: Host Specificity; Plasmids; Cloning, Molecular
- From:Malaysian Journal of Microbiology 2021;17(5):588-592
- CountryMalaysia
- Language:English
-
Abstract:
Aims:Expression of recombinant proteins across a range of different host organisms has profound contribution to the advancement in biotechnology. In this study, we aimed to construct a highly versatile broad host range (BHR) expression vector, designated as pYL101C.
Methodology and results:The Golden Gate cloning approach was used to construct pYL101C. Key features of pYL101C include a strong integron promoter (PINTc), a BHR pBBR1 origin of replication (ori), gentamycin resistance gene (GmR) as a selectable marker and a multiple cloning site (MCS) downstream of the promoter for easy-cloning purpose. To verify the functionality of pYL101C, we cloned the superfolder green fluorescent protein (sfGFP) reporter gene into pYL101C and transferred the resultant recombinant plasmid pYL101C::sfGFP into various Gram-negative bacteria. Transformants obtained stably expressed strong green fluorescence under blue light excitation even without selection after four passages.
Conclusion, significance and impact of study:The constructed BHR expression vector, pYL101C and recombinant pYL101C::sfGFP are stable and can be used to monitor the presence of Gram-negative bacteria, such as endophytes and pathogens in their hosts and environment. - Full text:20.2021my0059.pdf
