TY - JOUR
T1 - Engineering the bacterium Comamonas testosteroni CNB-1
T2 - Plasmid curing and genetic manipulation
AU - Tang, Qiang
AU - Lu, Ting
AU - Liu, Shuang Jiang
N1 - We are grateful to Dr. Nathan Hillson for providing the plasmid pKTTRFP. This work was supported by the National Science Foundation of China (NSFC grant No. 31230003 ) and a CAS/SAFEA international partnership program .
PY - 2018/5/15
Y1 - 2018/5/15
N2 - Comamonas species are attractive microbial hosts for bioremediation engineering due to their versatile metabolic capacity and diverse niches inhabited. To establish Comamonas testosteroni CNB-1—a common Comamonas strain—as a programmable cellular chassis, its indigenous 91-kb-long plasmid, pCNB1, must be eliminated; in addition, the organism has to exhibit feasiblity for effective genetic manipulation. Here we present a novel plasmid curing strategy involving rare-cutting homing endonuclease and a selection and counter-selection system. With a self-eliminating helper plasmid, we successfully removed the target plasmid pCNB1, along with the helper plasmid itself, in a single step at an efficiency of 64%. By enhancing the homing endonuclease expression, we were able to further improve the efficiency to almost 100%. The curing of pCNB1 provided a plasmid-free strain for hosting an IncP-type replicon based shuttle vehicle which can be utilized for gene circuit engineering. Additionally, using a PCR-based Cre-loxP system, we demonstrated an increased ability to genetically manipulate the chromosome of C. testosteroni CNB-1. This study provides a rapid and effective solution to eliminate indigenous plasmids, and also facilitates the development of C. testosteroni as a promising chassis for future remediation applications.
AB - Comamonas species are attractive microbial hosts for bioremediation engineering due to their versatile metabolic capacity and diverse niches inhabited. To establish Comamonas testosteroni CNB-1—a common Comamonas strain—as a programmable cellular chassis, its indigenous 91-kb-long plasmid, pCNB1, must be eliminated; in addition, the organism has to exhibit feasiblity for effective genetic manipulation. Here we present a novel plasmid curing strategy involving rare-cutting homing endonuclease and a selection and counter-selection system. With a self-eliminating helper plasmid, we successfully removed the target plasmid pCNB1, along with the helper plasmid itself, in a single step at an efficiency of 64%. By enhancing the homing endonuclease expression, we were able to further improve the efficiency to almost 100%. The curing of pCNB1 provided a plasmid-free strain for hosting an IncP-type replicon based shuttle vehicle which can be utilized for gene circuit engineering. Additionally, using a PCR-based Cre-loxP system, we demonstrated an increased ability to genetically manipulate the chromosome of C. testosteroni CNB-1. This study provides a rapid and effective solution to eliminate indigenous plasmids, and also facilitates the development of C. testosteroni as a promising chassis for future remediation applications.
KW - Bioremediation
KW - Comamonas testosteroni
KW - Cre-loxP
KW - Genome editing
KW - I-SceI
KW - Plasmid curing
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U2 - 10.1016/j.bej.2018.01.030
DO - 10.1016/j.bej.2018.01.030
M3 - Article
AN - SCOPUS:85041508626
SN - 1369-703X
VL - 133
SP - 74
EP - 82
JO - Biochemical Engineering Journal
JF - Biochemical Engineering Journal
ER -