Modulation of gene expression from the arabinose-inducible araBAD promoter A Khlebnikov, T Skaug and JD Keasling Department of Chemical Engineering, University of California, Berkeley, CA 94720 - 1462, USA The arabinose - inducible P BAD promoter suffers from all - or - none gene expression in which cells harboring the natively controlled arabinose transport gene ( araE ) are either induced or uninduced, the relative fraction of which is controlled by the concentration of arabinose. The population - averaged variation in expression from P BAD as a function of inducer concentration is proportional to the percentage of cells that are fully induced ( vs. uninduced ) rather than the level of expression in individual cells. Because of its undesirable effects on the expression of heterologous genes, the all - or - none phenomenon was eliminated in Escherichia coli by expression of araE from arabinose - independent ( either the Lactococcus lactis constitutive or IPTG - inducible lac ) promoters. In these arabinose - transport engineered cells, variation in P BAD expression with arabinose concentration was a result of variation of the expression level in individual cells with all cells in the population having approximately the same induction level. Journal of Industrial Microbiology & Biotechnology (2002) 29, 34 – 37 doi:10.1038/sj.jim.7000259 Keywords: arabinose; constitutive promoter; Escherichia coli; regulatable control; arabinose transport Introduction The arabinose - inducible araBAD promoter ( P BAD ) has been used extensively for expression of heterologous genes in Escherichia coli and other hosts [ 1,4,12,15,16,18 - 20 ]. In its simplest form, P BAD and the divergently transcribed araC, which encodes the AraC regulatory protein, are placed in an expression vector and transformed into a host capable of transporting arabinose into the cell [ 2 ]. When arabinose is added to the medium and transported into the cell, it binds to AraC, which in turn binds to the araI 1 and araI 2 sites between P BAD and P C [ 17 ]. In the absence of arabinose, the AraC represses transcription from P BAD and P C . This mechanism allows the araC - P BAD expression systems to provide: ( i ) a relatively low level of background expression in the absence of arabinose and ( ii ) modulation of the expression level by addition of L -arabinose. In a similar manner, AraC regulates transcription from the araE promoter ( P E ) and production of the low-affinity, high -capacity AraE arabinose transporter. The tight control offered by P BAD and P E results in a phenomenon ( all - or - none gene expression ) that may not be desirable for the expression of heterologous genes. When concentrations of arabinose intermediate between zero and that necessary for maximal induction are used to induce expression, a fraction of the cells become fully induced and the remainder are uninduced. One hypothesis for why this phenomenon occurs is that those cells that encounter inducer and are capable of transporting the inducer into the cell are able to induce expression from P BAD and P E , resulting in more AraE, more arabinose inside the cell, and even higher expression from P BAD and P E ; those cells that are not able to transport enough inducer to initiate expression from P BAD and P E remain uninduced. Recently, we demonstrated that expression of araE from an arabinose - independent promoter resulted in a homogeneous population of cells at all inducer concentrations and regulatable promoter control in each cell of the population [ 10 ]. In this paper we report on the development of several host / transporter systems that allow homogeneous expression from P BAD and tight control over gene expression. Materials and methods The bacteria and vectors used are summarized in Table 1. All DNA manipulations were performed in E. coli DH10B using established protocols [13] or as indicated below. Polymerase chain reaction ( PCR ) amplification of DNA was performed by using the Expand 2 high - fidelity PCR system ( Roche Molecular Biochemicals, Indianapolis, IN ) under conditions recommended by the manu- facturer. Sequencing and DNA oligonucleotide primer synthesis were preformed by Genemed Synthesis ( South San Francisco, CA ). The restriction digests and ligation reactions were performed as recommended by the restriction enzyme manufacturer ( Roche Molecular Biochemicals ). The ligated vectors were transformed into electrocompetent cells ( E. coli DH10B, E. coli CW2513 or E. coli CW2587 ) by electroporation ( field strength 18 kV / cm ) using a Bio - Rad E. coli Pulser 2 ( Bio - Rad, Hercules, CA ). Constitutive promoters of different strengths from Lactococcus lactis [ 7,8 ] were subcloned from the original vectors ( pCP8, pCP13, and pCP18 ) onto the broad - host - range, low-copy- number plasmid pJN105, containing the gentamycin resistance gene and the pBBR - 1 origin of replication [ 9 ]. The resulting plasmids were designated pJAT8, pJAT13, and pJAT18. The araE gene was amplified from genomic DNA of E. coli W3110 using PCR and placed on pJAT8, pJAT13, and pJAT18 resulting in plasmids pJAT8araE, pJAT13araE, and pJAT18araE, respectively [ 9 ] or on the medium - copy number vectors pMMB206 and Correspondence: Dr Jay D Keasling, Department of Chemical Engineering, University of California, Berkeley, CA 94720 - 1462, USA Received 20 November 2001; accepted 29 March 2002 Journal of Industrial Microbiology & Biotechnology (2002) 29, 34 – 37 D 2002 Nature Publishing Group All rights reserved 1367-5435/02 $25.00 www.nature.com / jim