Screening of promoters from rhizosphere metagenomic DNA using a promoter-trap vector and flow cytometric cell sorting.
We constructed a facilitative and efficient promoter-trap vector, pCM-EGFP, for capturing and analyzing functional promoters from environmental DNA. The pCM-EGFP vector showed good chloramphenicol sensitivity and no enhanced green fluorescent protein (EGFP) gene expression. Promoter libraries were constructed for screening promoters responding to naringenin, a key molecule released from plant roots. After electroporation, E. coli transformants were incubated in LB broth containing chloramphenicol (10 μg/ml) to select against transformants with no cloned promoter. E. coli cells were sorted using flow cytometry without naringenin, and then sorted again with high fluorescence after incubation in LB broth with naringenin (1 mM) at 28 °C for 12 h. The inducible properties of approximately 400 sorted cells were evaluated, with most cells showing only strong EGFP gene expression without inducible properties. Two clones (5-4E and 15-3D) displayed naringenin inducibility, and both contained a promoter bounded by a TetR-family regulator. The regulator knock-out mutant of the 5-4E clone lost its ability to be induced by naringenin. In conclusion, the pCM-EGFP vector may be used as an efficient promoter-trap vector and a combination of the vector with flow cytometric cell sorting was demonstrated to be an useful method for screening promoters responding to specific conditions or inducers.
Department of Life Science and Research Center for Biomolecules and Biosystems, Chung-Ang University, Seoul, Republic of Korea.
Se Hee
SH
Lee
Jeong Myeong
JM
Kim
Hyo Jung
HJ
Lee
Che Ok
CO
Jeon
D003470:Culture Media; D004247:DNA / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D044950:Flavanones / Q000378:metabolism; D005434:Flow Cytometry; D015723:Gene Library; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D054892:Metagenome; D056186:Metagenomics; D011401:Promoter Regions, Genetic; D058441:Rhizosphere; D012988:Soil Microbiology
D003470:Culture Media; D044950:Flavanones; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA; C005273:naringenin