Design of Synthetic Promoters for Gene Circuits in Mammalian Cells

Synthetic mRNAs, which are produced by in vitro transcription, have been recently attracting attention because they can express any transgenes without the risk of insertional mutagenesis. Although current synthetic mRNA medicine is not designed for spatiotemporal or cell-selective regulation, many preclinical studies have developed the systems for the translational regulation of synthetic mRNAs. Such translational regulation systems will cope with high efficacy and low adverse effects by producing the appropriate amount of therapeutic proteins, depending on the context. Protein-based regulation is one of the most promising approaches for the translational regulation of synthetic mRNAs. As synthetic mRNAs can encode not only output proteins but also regulator proteins, all components of protein-based regulation systems can be delivered as synthetic mRNAs. In addition, in the protein-based regulation systems, the output protein can be utilized as the input for the subsequent regulation to construct multi-layered gene circuits, which enable complex and sophisticated regulation. In this review, I introduce what types of proteins have been used for translational regulation, how to combine them, and how to design effective gene circuits.

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Synthetic therapeutic gene circuits in mammalian cells

FEBS Letters ◽

10.1016/j.febslet.2014.05.003 ◽

2014 ◽

Vol 588 (15) ◽

pp. 2537-2544 ◽

Cited By ~ 47

Author(s):

Haifeng Ye ◽

Martin Fussenegger

Keyword(s):

Mammalian Cells ◽

Therapeutic Gene ◽

Gene Circuits

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Synthetic Gene Circuits for Antimicrobial Resistance and Cancer Research

10.5772/intechopen.99329 ◽

2021 ◽

Author(s):

Kevin S. Farquhar ◽

Michael Tyler Guinn ◽

Gábor Balázsi ◽

Daniel A. Charlebois

Keyword(s):

Mammalian Cells ◽

Rational Design ◽

Study Drug ◽

Quantitative Model ◽

Synthetic Gene ◽

Modeling Tools ◽

Gene Circuits ◽

Novel Treatments ◽

Synthetic Gene Circuits ◽

Temporal Dimensions

Mathematical models and synthetic gene circuits are powerful tools to develop novel treatments for patients with drug-resistant infections and cancers. Mathematical modeling guides the rational design of synthetic gene circuits. These systems are then assembled into unified constructs from existing and/or modified genetic components from a range of organisms. In this chapter, we describe modeling tools for the design and characterization of chemical- and light-inducible synthetic gene circuits in different organisms and highlight how synthetic gene circuits are advancing biomedical research. Specifically, we demonstrate how these quantitative model systems are being used to study drug resistance in microbes and to probe the spatial–temporal dimensions of cancer in mammalian cells.

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Screening of CHO-K1 endogenous promoters for expressing recombinant proteins in mammalian cell cultures

10.1101/2021.10.13.464282 ◽

2021 ◽

Author(s):

Ileana Tossolini ◽

Agustina Gugliotta ◽

Fernando Lopez Diaz ◽

Ricardo Kratje ◽

Claudio Prieto

Keyword(s):

Recombinant Proteins ◽

Mammalian Cells ◽

High Strength ◽

High Rate ◽

High Expression ◽

Expression Vectors ◽

Cmv Promoter ◽

Promising Alternative ◽

Expression Levels ◽

Synthetic Promoters

For the production of recombinant protein therapeutics in mammalian cells, a high rate of gene expression is desired and hence strong viral-derived promoters are commonly used. However, they usually induce cellular stress and can be susceptible to epigenetic silencing. Endogenous promoters, which coordinates their activity with cellular and bioprocess dynamics while at the same time they maintain high expression levels, may help to avoid such drawbacks. In this work, endogenous promoters were identified based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density. The promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new expression vectors for the production of recombinant proteins in mammalian cells. The in silico derived promoter regions were experimentally verified and the majority showed transcriptional activity comparable or higher than CMV. Also, stable expression following a reduction of culture temperature was investigated. The characterized endogenous promoters (excluding Rps18) constitute a promising alternative to CMV promoter due to their high strength, long-term expression stability and integration into the regulatory network of the host cell. These promoters may also comprise an initial panel for designing cell engineering strategies and synthetic promoters, as well as for industrial cell line development.

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Functional Characterization of Insulation Effect for Synthetic Gene Circuits in Mammalian Cells

ACS Synthetic Biology ◽

10.1021/acssynbio.7b00134 ◽

2018 ◽

Vol 7 (2) ◽

pp. 412-418 ◽

Cited By ~ 2

Author(s):

Weixi Liao ◽

Bing Liu ◽

Chih-Chun Chang ◽

Ling-Jun Lin ◽

Che Lin ◽

...

Keyword(s):

Mammalian Cells ◽

Functional Characterization ◽

Synthetic Gene ◽

Gene Circuits ◽

Synthetic Gene Circuits ◽

Insulation Effect

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Noise-reducing optogenetic negative-feedback gene circuits in human cells

Nucleic Acids Research ◽

10.1093/nar/gkz556 ◽

2019 ◽

Vol 47 (14) ◽

pp. 7703-7714 ◽

Cited By ~ 11

Author(s):

Michael Tyler Guinn ◽

Gábor Balázsi

Keyword(s):

Gene Expression ◽

Noise Reduction ◽

Negative Feedback ◽

Mammalian Cells ◽

Cellular Proliferation ◽

Protein Domain ◽

Gene Circuits ◽

Expression Control ◽

Gene Expression Control

Abstract Gene autorepression is widely present in nature and is also employed in synthetic biology, partly to reduce gene expression noise in cells. Optogenetic systems have recently been developed for controlling gene expression levels in mammalian cells, but most have utilized activator-based proteins, neglecting negative feedback except for in silico control. Here, we engineer optogenetic gene circuits into mammalian cells to achieve noise-reduction for precise gene expression control by genetic, in vitro negative feedback. We build a toolset of these noise-reducing Light-Inducible Tuner (LITer) gene circuits using the TetR repressor fused with a Tet-inhibiting peptide (TIP) or a degradation tag through the light-sensitive LOV2 protein domain. These LITers provide a range of nearly 4-fold gene expression control and up to 5-fold noise reduction from existing optogenetic systems. Moreover, we use the LITer gene circuit architecture to control gene expression of the cancer oncogene KRAS(G12V) and study its downstream effects through phospho-ERK levels and cellular proliferation. Overall, these novel LITer optogenetic platforms should enable precise spatiotemporal perturbations for studying multicellular phenotypes in developmental biology, oncology and other biomedical fields of research.

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Engineering orthogonal dual transcription factors for multi-input synthetic promoters

Nature Communications ◽

10.1038/ncomms13858 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 24

Author(s):

Andreas K. Brödel ◽

Alfonso Jaramillo ◽

Mark Isalan

Keyword(s):

Transcription Factors ◽

Synthetic Biology ◽

Directed Evolution ◽

Logic Gates ◽

Bacteriophage Λ ◽

Gene Circuits ◽

Synthetic Promoters ◽

Core Set ◽

Artificial Gene ◽

Current Engineering

Abstract 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.

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Saccharomyces cerevisiae Promoter Engineering before and during the Synthetic Biology Era

Biology ◽

10.3390/biology10060504 ◽

2021 ◽

Vol 10 (6) ◽

pp. 504

Author(s):

Xiaofan Feng ◽

Mario Andrea Marchisio

Keyword(s):

Synthetic Biology ◽

Dna Sequences ◽

Genetic Circuits ◽

Gene Circuits ◽

Promoter Engineering ◽

Rna Molecules ◽

Promoter Sequences ◽

Synthetic Promoters ◽

Synthetic Gene Circuits ◽

Wet Lab

Synthetic gene circuits are made of DNA sequences, referred to as transcription units, that communicate by exchanging proteins or RNA molecules. Proteins are, mostly, transcription factors that bind promoter sequences to modulate the expression of other molecules. Promoters are, therefore, key components in genetic circuits. In this review, we focus our attention on the construction of artificial promoters for the yeast S. cerevisiae, a popular chassis for gene circuits. We describe the initial techniques and achievements in promoter engineering that predated the start of the Synthetic Biology epoch of about 20 years. We present the main applications of synthetic promoters built via different methods and discuss the latest innovations in the wet-lab engineering of novel promoter sequences.

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DNA sequence requirements for transcriptional initiator activity in mammalian cells

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.116-127.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 116-127

Author(s):

R Javahery ◽

A Khachi ◽

K Lo ◽

B Zenzie-Gregory ◽

S T Smale

Keyword(s):

Dna Sequence ◽

Dna Sequences ◽

Mammalian Cells ◽

Binding Protein ◽

Consensus Sequence ◽

Tata Box ◽

Start Site ◽

Sequence Element ◽

Synthetic Promoters ◽

Sequence Requirements

A transcriptional initiator (Inr) for mammalian RNA polymerase II can be defined as a DNA sequence element that overlaps a transcription start site and is sufficient for (i) determining the start site location in a promoter that lacks a TATA box and (ii) enhancing the strength of a promoter that contains a TATA box. We have prepared synthetic promoters containing random nucleotides downstream of Sp1 binding sites to determine the range of DNA sequences that convey Inr activity. Numerous sequences behaved as functional Inrs in an in vitro transcription assay, but the Inr activities varied dramatically. An examination of the functional elements revealed loose but consistent sequence requirements, with the approximate consensus sequence Py Py A+1 N T/A Py Py. Most importantly, almost every functional Inr that has been described fits into the consensus sequence that we have defined. Although several proteins have been reported to bind to specific Inrs, manipulation of those elements failed to correlate protein binding with Inr activity. The simplest model to explain these results is that all or most Inrs are recognized by a universal binding protein, similar to the functional recognition of all TATA sequences by the same TATA-binding protein. The previously reported proteins that bind near specific Inr elements may augment the strength of an Inr or may impart transcriptional regulation through an Inr.

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