Engineering orthogonal dual transcription factors for multi-input synthetic promoters

Synthetic biology has seen an explosive growth in the capability of engineering artificial gene circuits from transcription factors (TFs), particularly in bacteria. However, most artificial networks still employ the same core set of TFs (for example LacI, TetR and cI). The TFs mostly function via repression and it is difficult to integrate multiple inputs in promoter logic. Here we present to our knowledge the first set of dual activator-repressor switches for orthogonal logic gates, based on bacteriophage λ cI variants and multi-input promoter architectures. Our toolkit contains 12 TFs, flexibly operating as activators, repressors, dual activator–repressors or dual repressor–repressors, on up to 270 synthetic promoters. To engineer non cross-reacting cI variants, we design a new M13 phagemid-based system for the directed evolution of biomolecules. Because cI is used in so many synthetic biology projects, the new set of variants will easily slot into the existing projects of other groups, greatly expanding current engineering capacities

CHO genome mining for synthetic promoter design

Synthetic promoters are an attractive alternative for use in mammalian hosts such as CHO cells as they can be designed de novo with user-defined functionalities. In this study, we describe and validate a method for bioprocess-directed design of synthetic promoters utilizing CHO genomic sequence information. We designed promoters with two objective features, (i) constitutive high-level recombinant gene transcription, and (ii) upregulated transcription under mild hypothermia or late-stage culture. CHO genes varying in transcriptional activity were selected based on a comparative analysis of RNA-Seq transcript levels in normal and biphasic cultures in combination with estimates of mRNA half-life from published genome scale datasets. Discrete transcription factor regulatory elements (TFREs) upstream of these genes were informatically identified and functionally screened in vitro to identify a subset of TFREs with the potential to support high activity recombinant gene transcription during biphasic cell culture processes. Two libraries of heterotypic synthetic promoters with varying TFRE combinations were then designed in silico that exhibited a maximal 2.5-fold increase in transcriptional strength over the CMV-IE promoter after transient transfection into host CHO-K1 cells. A subset of synthetic promoters was then used to create stable transfectant pools using CHO-K1 cells under glutamine synthetase selection. Whilst not achieving the maximal 2.5-fold increase in productivity over stable pools harboring the CMV promoter, all stably transfected cells utilizing synthetic promoters exhibited increased reporter production - up to 1.6-fold that of cells employing CMV, both in the presence or absence of intron A immediately downstream of the promoter. The increased productivity of stably transfected cells harboring synthetic promoters was maintained during fed-batch culture, with or without a transition to mild hypothermia at the onset of stationary phase. Our data exemplify that it is important to consider both host cell and intended bioprocess contexts as design criteria in the de novo construction of synthetic genetic parts for mammalian cell engineering

TATA is a modular component of synthetic promoters

The expression of most genes is regulated by multiple transcription factors. The interactions between transcription factors produce complex patterns of gene expression that are not always obvious from the arrangement of cis-regulatory elements in a promoter. One critical element of promoters is the TATA box, the docking site for the RNA polymerase holoenzyme. Using a synthetic promoter system coupled to a thermodynamic model of combinatorial regulation, we analyze the effects of different strength TATA boxes on various aspects of combinatorial cis-regulation. The thermodynamic model explains 75% of the variance in gene expression in synthetic promoter libraries with different strength TATA boxes, suggesting that many of the salient aspects of cis-regulation are captured by the model. Our results demonstrate that the effect of changing the TATA box on gene expression is the same for all synthetic promoters regardless of the arrangement of cis-regulatory sites we studied. Our analysis also showed that in our synthetic system the strength of the RNA polymerase–TATA interaction does not alter the combinatorial interactions between transcription factors, or between transcription factors and RNA polymerase. Finally, we show that although stronger TATA boxes increase expression in a predictable fashion, stronger TATA boxes have very little effect on noise in our synthetic promoters, regardless of the arrangement of cis-regulatory sites. Our results support a modular model of promoter function, where cis-regulatory elements can be mixed and matched (programmed) with outcomes on expression that are predictable based on the rules of simple protein–protein and protein–DNA interactions

Environment-specific combinatorial cis-regulation in synthetic promoters

When a cell's environment changes, a large transcriptional response often takes place. The exquisite sensitivity and specificity of these responses are controlled in large part by the combinations of cis-regulatory elements that reside in gene promoters and adjacent control regions. Here, we present a study aimed at accurately modeling the relationship between combinations of cis-regulatory elements and the expression levels they drive in different environments. We constructed four libraries of synthetic promoters in yeast, consisting of combinations of transcription factor binding sites and assayed their expression in four different environments. Thermodynamic models relating promoter sequences to their corresponding four expression levels explained at least 56% of the variation in expression in each library through the different conditions. Analyses of these models suggested that a large fraction of regulated gene expression is explained by changes in the effective concentration of sequence-specific transcription factors, and we show that in most cases, the corresponding transcription factors are expressed in a pattern that is predicted by the thermodynamic models. Our analysis uncovered two binding sites that switch from activators to repressors in different environmental conditions. In both the cases, the switch was not the result of a single transcription factor changing regulatory modes, but most likely due to competition between multiple factors binding to the same site. Our analysis suggests that this mode of regulation allows for large and steep changes in expression in response to changing transcription factor concentrations. Our results demonstrate that many complex changes in gene expression are accurately explained by simple changes in the effective concentrations of transcription factors

Ethylene Response of Plum ACC Synthase 1 (ACS1) Promoter is Mediated through the Binding Site of Abscisic Acid Insensitive 5 (ABI5).

The enzyme 1-amino-cyclopropane-1-carboxylic acid synthase (ACS) participates in the ethylene biosynthesis pathways and it is tightly regulated transcriptionally and post-translationally. Notwithstanding its major role in climacteric fruit ripening, the transcriptional regulation of ACS during ripening is not fully understood. We studied fruit ripening in two Japanese plum cultivars, the climacteric Santa Rosa (SR) and its non-climacteric bud sport mutant, Sweet Miriam (SM). As the two cultivars show considerable difference in ACS expression, they provide a good system for the study of the transcriptional regulation of the gene. To investigate the differential transcriptional regulation of ACS1 genes in the SR and SM, their promoter region, which showed only minor sequence differences, was isolated and used to identify the binding of transcription factors interacting with specific ACS1 cis-acting elements. Three transcription factors (TFs), abscisic acid-insensitive 5 (ABI5), GLABRA 2 (GL2), and TCP2, showed specific binding to the ACS1 promoter. Synthetic DNA fragments containing multiple cis-acting elements of these TFs fused to β-glucuronidase (GUS), showed the ABI5 binding site mediated ethylene and abscisic acid (ABA) responses of the promoter. While TCP2 and GL2 showed constant and similar expression levels in SM and SR fruit during ripening, ABI5 expression in SM fruits was lower than in SR fruits during advanced fruit ripening states. Overall, the work demonstrates the complex transcriptional regulation of ACS1

Automated design of bacterial genome sequences

Background: Organisms have evolved ways of regulating transcription to better adapt to varying environments. Could the current functional genomics data and models support the possibility of engineering a genome with completely rearranged gene organization while the cell maintains its behavior under environmental challenges? How would we proceed to design a full nucleotide sequence for such genomes? Results: As a first step towards answering such questions, recent work showed that it is possible to design alternative transcriptomic models showing the same behavior under environmental variations than the wild-type model. A second step would require providing evidence that it is possible to provide a nucleotide sequence for a genome encoding such transcriptional model. We used computational design techniques to design a rewired global transcriptional regulation of Escherichia coli, yet showing a similar transcriptomic response than the wild-type. Afterwards, we “compiled” the transcriptional networks into nucleotide sequences to obtain the final genome sequence. Our computational evolution procedure ensures that we can maintain the genotype-phenotype mapping during the rewiring of the regulatory network. We found that it is theoretically possible to reorganize E. coli genome into 86% fewer regulated operons. Such refactored genomes are constituted by operons that contain sets of genes sharing around the 60% of their biological functions and, if evolved under highly variable environmental conditions, have regulatory networks, which turn out to respond more than 20% faster to multiple external perturbations. Conclusions: This work provides the first algorithm for producing a genome sequence encoding a rewired transcriptional regulation with wild-type behavior under alternative environments

An Experimental Framework to Examine the Influence of Promoter Architecture and Genomic Context on Gene Expression

Transcription is a fundamental process of gene expression. Information stored in DNA is transcribed into different types of mobile RNA, which play a role in various essential processes of the cell, e.g. translation. However, cells do not need all the information stored in their DNA at the same time. Therefore, the process of transcription gets regulated by a plethora of mechanisms. One frequently discussed but poorly understood mechanism of transcription regulation is DNA supercoiling [Travers and Muskhelishvili, 2005]. Whereby, the process of transcription itself affects the DNA-topology up- and downstream of the transcription machinery as described in the twin supercoiling domain model [Liu and Wang, 1987]. This phenomenon is called Transcription Coupled DNA Supercoiling (TCDS). It has also been shown that genes react individually to changes in DNA supercoiling and that there is a selection pressure on adapting to the DNA supercoiling levels emitted by neighbouring gene expression [Sobetzko, 2016]. The system in which promoters react to changes in DNA supercoiling is as diverse as there are promoters; notably, some promoters seem not to respond to DNA supercoiling at all. Thus, this raises the question as to which elements within different promoter types cause them to respond to TCDS so differently. In this thesis, I built a pipeline to investigate the effects of TCDS and DNA supercoiling on promoters. Firstly, I created a plasmid toolbox, which allows modular assembly of transcription units. The central feature of this toolbox is the flexibility to test different arrangements of multiple transcription units. I achieved this by adapting the well established Modular Cloning (MoClo) standard [Weber et al., 2011] and build my toolbox around it. I thus created a system that works on both its own and is compatible with the existing standard MoClo protocol. In the second part of this thesis, I established an experimental pipeline using synthetic σ70-promoters to investigate the influence of DNA supercoiling on transcription. The experimental setup allowed precise changes in parts of the promoter and at the same time created a library of these promoters. Using this pipeline to investigate the spacer region of the promoter, I was able to confirm that the spacer influences the promoter strength. Further, I showed that the promoter spacer has only a limited effect on the supercoiling sensitivity of a promoter. I also showed that a 5‘-TGTG-3‘ motif in the spacer region could lower transcription by enhancing RNA-polymerase (RNAP)-binding. Moreover, the experimental setup also showed the constraints of using the DNA-relaxing drug novobiocin on a plasmid-based system. Hence, to further investigate the effects of TCDS on neighbouring transcription, I applied an optogenetically-controllable promoter to the previously established pipeline. Finally, I began to explore the possibility of integrating my experimental promoter setup into any genomic position. As such, a CRISPR/Cas9-based homologous re-combination system was developed further to make it modular and compatible with the Modular Cloning protocol. I could show the first features of this system to work