Development of High-Performance Whole Cell Biosensors Aided by Statistical Modeling

Whole cell biosensors are genetic systems that link the presence of a chemical, or other stimulus, to a user-defined gene expression output for applications in sensing and control. However, the gene expression level of biosensor regulatory components required for optimal performance is nonintuitive, and classical iterative approaches do not efficiently explore multidimensional experimental space. To overcome these challenges, we used a design of experiments (DoE) methodology to efficiently map gene expression levels and provide biosensors with enhanced performance. This methodology was applied to two biosensors that respond to catabolic breakdown products of lignin biomass, protocatechuic acid and ferulic acid. Utilizing DoE we systematically modified biosensor dose–response behavior by increasing the maximum signal output (up to 30-fold increase), improving dynamic range (>500-fold), expanding the sensing range (∼4-orders of magnitude), increasing sensitivity (by >1500-fold), and modulated the slope of the curve to afford biosensors designs with both digital and analogue dose–response behavior. This DoE method shows promise for the optimization of regulatory systems and metabolic pathways constructed from novel, poorly characterized parts.


Additional methods -Molecular cloning
The PCA biosensor was assembled by isothermal assembly from the following fragments: pSEVA131 linearized by inverse PCR (primers AB9/10); mCherry amplified with primers AB15/28 from a synthetic gene (GeneArt); sfGFP amplified with primers AB18/27 from a synthetic gene (GeneArt), and synthetic DNA (IDT) incorporating the ProB promoter (1) fused to a strong RBS (gaaataaggaggtaatacaa) (2), the P PV promoter (3) fused to the G10 RBS (4) and a 150 bp spacer (5) to yield the template plasmid (p131B). Promoter (P reg -lib and P out -lib) and RBS (RBS out -lib) libraries were generated by linearising p131B by inverse PCR with primers AB27/94 (for P out -lib and RBS out -lib) and AB146/147 (for P reg -lib) and inserting the following degenerate ssDNA oligonucleotides via isothermal assembly: for P out -lib oligo AB115, for RBS out -lib oligo AB114, and for P reg -lib oligo AB148 (Supplementary Table 12). The library members were designated p131B-BX for P reg -lib, p131B-GX for RBS out -lib, and p131-VX for P out -lib, with X denoting the clone number, which was assigned based on subsequent screening and rank order of expression output.
For the ferulic acid biosensor (FAB) designs, the pFABsP vector was constructed by isothermal assembly, using (i) pET28a (Novagen) served as a backbone and linearized by PCR with primers FAB1/2 to remove lacI and the T7 promoter; (ii) the chimeric P LC promoteroperator (7) and the G10 RBS were incorporated into the forward primer of the FAB3/4 pair and used to amplify sfGFP from a synthetic gene (IDT) and (iii) the FerC transcription factor and FerA enzyme (7) amplified with primers FAB5/6 from p15FABs to yield pFABsP. The new strong promoter-operator P LC2 (Supplementary Figure 3) was synthesised as a gBlock (IDT) and exchanged with the P LC promoter by isothermal assembly using pFABsP LC linearized by inverse PCR with primers FAB6/7.
Forward primers (FAB21, FAB22, FAB23) were designed with the sequences from the RBS out library corresponding to levels 0.81, 0.89 and 0.94. A reverse primer (FAB24) with overlapping nucleotides to the forward primers was designed. Inverse PCR of pFABs9 with these primers followed by isothermal assembly was carried out to insert the new RBS sequences.
The pcaK gene from Pseudomonas putida was synthetized (IDT) with codon-optimsation for expression in E. coli with a short translational initiation region (AGGAGGAAAAAAA) at the 5' of the start codon. The gene was inserted downstream of pcaV via in vivo assembly into plasmid p131C-B10, linearised by PCR with primers AB10/167, to create p131C-B10-pcaK.
The extender plasmid p261-lacI-pcaK, contains the p15A origin and a kanamycin selection marker, and was assembled by isothermal assembly from the following fragments: (i) the  The titration was carried out with a PCA concentration ranging from to 0.0128 to 1000 µM.
OFF and ON measurements were made in the absence or presence of 1 mM PCA, respectively. The values for OFF, ON and OFF/ON indicate the mean of three biological replicates with ± denoting the standard deviation of those replicates.