Directed Evolution of AraC for Improved Compatibility of Arabinose- and Lactose-Inducible Promoters

Abstract

Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (P BAD ) system that is more compatible with IPTG (isopropyl-␤-D-1-thiogalactopyranoside) induction of a lactose-inducible (P lac ) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.Recent advances in metabolic engineering and synthetic biology have increased the use of multiple genes (17) for various applications, such as the production of medicines (22,24) and the construction of complex genetic circuits (21, 30). Many genetic circuits, such as inverters (32), logic gates (8), pulse generators (4), band-pass filters, and oscillators (12), have been used to develop strains of bacteria that can communicate to form two-dimensional patterns, control their population density, and attack tumor cells in response to environmental cues (1,3,33). It is often necessary to introduce multiple genes and control the expression levels of those genes independently in order to accomplish these tasks. To date, a number of inducible promoters have been developed for Escherichia coli and other bacteria (2, 11, 16) which, in theory, should make it possible to independently control the expression of more than one gene. However, some pairs of promoters suffer from cross talk (an inducer of one promoter affects the expression from another promoter), making it difficult to simultaneously and independently control the expression levels of multiple genes. In contrast to electrical systems in which system interactions are well characterized and can be isolated from one another, it is often difficult or impossible to isolate biological components from activation or inhibition by other components in the cell.One example of cross talk involves a system containing two of the most useful promoters for gene expression in bacteria: the IPTG (isopropyl-␤-D-1-thiogalactopyranoside)-inducible lac promoter (P lac ) (15) and the arabinose-inducible araBAD promoter (P BAD ) (13, 29). The araBAD promoter system is regulated by the transcriptional regulator AraC. In the absence of arabinose, an AraC dimer creates a 210-base-pair DNA loop by contacting two widely separated half-sites (I 1 and O 2 ) on the DNA and represses transcription from P BAD (19,20,28). The binding of arabinose to AraC changes the position of the AraC dimer, causing the protein to preferentially bind different DNA-binding half-sites (I 1 and I 2 ). The change in configuration causes the dimer to release the DNA loop and allow transcription from P BAD (14,19,28).AraC is a 292-residue protein consisting of an N-terminal domain (residues 1 to 170) joined to a C-terminal DNA-b...

Identification of Transport Proteins Involved in Free Fatty Acid Efflux in Escherichia coli

“…Plasmid pBAD33* is a modified version of pBAD33 (44) harboring a mutant form of araC with cysteine 280 converted to a premature stop codon (AraC-C280*). This mutation has been previously observed to reduce inhibition of gene expression from the P BAD promoter in the presence of IPTG (45), allowing the use of both inducing agents simultaneously. The plasmid was generated by PCR using primers 51 and 52 with template pBAD33, which introduced the C280* mutation and an XhoI restriction site at the 5= and 3= ends.…”

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“…In addition, IPTG is known to be an inhibitor of the araBADp expression system. Crosstalk between araBADp and lac operator-controlled promoters prevents them from being used simultaneously in the same cell (53). Under the time-delayed in vivo assembly conditions, this inhibitory effect of IPTG was advantageous, since the repression of the araBAD promoter was further strengthened.…”

Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels

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