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. 2020 Jun 12:8:551.
doi: 10.3389/fbioe.2020.00551. eCollection 2020.

Characterization of Context-Dependent Effects on Synthetic Promoters

Sebastian Köbbing  1 , Lars M Blank  1 , Nick Wierckx  1   2
Affiliations

Characterization of Context-Dependent Effects on Synthetic Promoters

Sebastian Köbbing et al. Front Bioeng Biotechnol. .

Abstract

Understanding the composability of genetic elements is central to synthetic biology. Even for seemingly well-known elements such as a sigma 70 promoter the genetic context-dependent variability of promoter activity remains poorly understood. The lack of understanding of sequence to function results in highly limited de novo design of novel genetic element combinations. To address this issue, we characterized in detail concatenated "stacked" synthetic promoters including varying spacer sequence lengths and compared the transcription strength to the output of the individual promoters. The proxy for promoter activity, the msfGFP synthesis from stacked promoters was consistently lower than expected from the sum of the activities of the single promoters. While the spacer sequence itself had no activity, it drastically affected promoter activities when placed up- or downstream of a promoter. Single promoter-spacer combinations revealed a bivalent effect on msfGFP synthesis. By systematic analysis of promoter and spacer combinations, a semi-empirical correlation was developed to determine the combined activity of stacked promoters.

Keywords: Pseudomonas putida; Tn7 transposon; heterologous expression; synthetic biology; synthetic promoter libraries; tandem promoter.

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Figures

Figure 1
Figure 1
Identification of the optimal spacer length between the two promoters 14f and 14g using a mini Tn7 vector (Zobel et al., 2015). (A) Two PCR reactions were performed to generate stacked promoters with longer (>45 bp) spacer sequences and promoter combinations with the 80i spacer sequence. In a first PCR reaction long single stranded DNA oligonucleotides containing one promoter sequence (yellow or blue) were annealed via complementary sequences in the spacer (gray) and extended by Q5 polymerase to double stranded DNA. The resulting dsDNA fragment was amplified in a second PCR. (B) Structural organization of stacked promoters and of the mini Tn7 used in this study after genomic integration. Stacked promoters consisting of promoter sequences at two positions separated by a spacer were inserted via restriction sites PacI and AvrII. BCD2 element for translational coupling and msfGFP as reporter gene. Tn7 module contains a GmR marker for selection and two terminators (T0 and T1) for insulation of the probe. Tn7R and Tn7L are recognized by a transposase. (C) Tested spacers contained between 10 and 100 bp. P. putida KT2440 attTn7::BGf##g-mfsGFP, where ## refers to the number of nucleotides in the spacer sequence (gray bars), were cultured in a BioLector in minimal medium with 20 mM glucose in a 96 well plate. The control strains BG13 with the Pem7 promoter of average strength, the individual promoters 14f and 14g, and promoterless BG and wild type P. putida KT2440, as well as additional controls with two 80 bp spacers 80i and 80new are also shown. Identical strains from at least two different transformations were tested, with three biological replicates each. Error bars indicate the standard error of the mean (n > 6).
Figure 2
Figure 2
Characterization of context-depended promoter activities using the 80i spacer. Promoters with both up- and downstream spacer were tested. Shown are promoter activities for the original promoters (dark bars) from Zobel et al. (2015) and promoter-spacer (14x_80i) as well as spacer-promoter (80i_14x) combinations (gray bars), where x stands for promoter 14a to 14g. All constructs were genomically integrated in P. putida KT2440. Strains were cultured in a BioLector in minimal medium with 20 mM glucose in a 96 well plate Identical strains from at least two different transformations were tested, with three biological replicates each. Vertical dotted lines are separating individual sets. Error bars indicate the standard error of the mean (n > 6).
Figure 3
Figure 3
Comparison of experimental values, context-specific and context-unspecific prediction of promoter activities for stacked promoter with the 80i spacer separating promoters 14a to 14g. (A) Shown are determined promoter activities for stacked promoters (light gray bars), context-specific activities calculated with context-depended values (gray bars) and context-unspecific activities using original promoter activities (black bars). All constructs were genomically integrated into P. putida KT2440. Cultivation was done in a BioLector in minimal medium with 20 mM glucose in a 96 well plate. Identical strains from at least three different transformations were tested, with three biological replicates each. Error bars indicate the standard error of the mean (n > 9). (B) Coefficient of variation (CV) of context-specific (light gray bars) and context-unspecific (black bars) prediction of resulting promoter activities. (C) Plot of msfGFP transcription levels normalized to rpoD determined by quantitative real time PCR (qRT-PCR) and promoter activities from single promoter controls and stacked promoters. Wild type P. putida KT2440 was used as negative control. All constructs are genomically integrated into the genome of P. putida KT2440. Cultivations to determine promoter activities were done in a BioLector in minimal medium with 20 mM glucose in a 96 well plate. Cultivation to determine transcription levels was done in 24 well System Duetz plates containing minimal medium with 20 mM glucose. Identical strains from at least two different transformations were tested, with three biological replicates each. Error bars indicate the standard error of the mean (n > 6).
Figure 4
Figure 4
Comparison of screened and characterized promoter sequences based on 14g with single nucleotide polymorphisms. (A) Original promoter sequence of 14g with highlighted−35 and−10 elements (in bold). Restriction sites PacI and AvrII are underlined and positions of the modified core promoter sequence are given with numbers above the sequence. (B) msfGFP fluorescence of E. coli PIR2 bearing plasmid pBG14g with degenerate bases at 30 positions along the core promoter sequence. Changed position is shown on the x axis. Determined values are ranked by fluorescence intensity of 14 strains tested for each position. Strains were cultivated in 96 well System Duetz plates with LB medium supplemented with 50 mg L−1 kanamycin. Fluorescence and optical density were measured with a plate reader. The dotted line indicates the promoter activity of the 14g control. (C) Chosen SNP promoter constructs were genomically integrated into P. putida KT2440. Strains are named 14G_##n, whereas ## stands for the position in the promoter sequence and n for a nucleotide (A, C, G or T). Cultivation was done in a BioLector in minimal medium with 20 mM glucose in 96 well plates. Identical strains from at least two different transformations were tested, with three biological replicates each. Error bars indicate the standard error of the mean (n > 6).
Figure 5
Figure 5
Characterization of the effect of the 80i spacer on a single nucleotide exchange promoter library based on promoter 14g genomically integrated in P. putida KT2440. (A) Promoter activities derived from GFP fluorescence analysis of SNP promoters with (gray bars) and without (black bars) downstream 80i spacer. Cultivation was done in a BioLector in minimal medium with 20 mM glucose in 96 well plates. Identical strains from at least two different transformations were tested, with three biological replicates each. Vertical dotted lines are separating individual sets. Error bars indicate the standard error of the mean (n > 6). (B) Box and whiskers plot of the relative effect of the downstream 80i spacer on promoter activities of the SNP library and the original calibrated promoter library from Zobel et al. (2015). The effect of the spacer is calculated as the % change of the promoter with downstream 80i sequence compared to the original promoter. Data points are indicated in red.
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