Evolutionary Design of Choline-Inducible and -Repressible T7-Based Induction Systems

Ike, Kohei; Arasawa, Yusuke; Koizumi, Satoshi; Mihashi, S; Kawai-Noma, Shigeko; Saito, Kyoichi; Umeno, Daisuke

doi:10.1021/acssynbio.5b00107

Cited by 24 publications

(36 citation statements)

References 65 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Numerous successful efforts have been made to engineer the T7 RNAP to recognize different promoter sequences ( 38–42 ) as well as to generally enhance its activity ( 31 , 32 ). Further, T7 RNAP has been split to generate inducible ( 43 , 44 ) and logic gated operations ( 45–47 ). Combined, these efforts yield an impressive library of T7 RNAPs that can target orthogonal promoters with very high transcriptional activation.…”

Section: Introductionmentioning

confidence: 99%

Programmable T7-based synthetic transcription factors

Hussey

McMillen

2018

Nucleic Acids Research

View full text Add to dashboard Cite

Despite recent progress on synthetic transcription factor generation in eukaryotes, there remains a need for high-activity bacterial versions of these systems. In synthetic biology applications, it is useful for transcription factors to have two key features: they should be orthogonal (influencing only their own targets, with minimal off-target effects), and programmable (able to be directed to a wide range of user-specified transcriptional start sites). The RNA polymerase of the bacteriophage T7 has a number of appealing properties for synthetic biological designs: it can produce high transcription rates; it is a compact, single-subunit polymerase that has been functionally expressed in a variety of organisms; and its viral origin reduces the connection between its activity and that of its host's transcriptional machinery. We have created a system where a T7 RNA polymerase is recruited to transcriptional start sites by DNA binding proteins, either directly or bridged through protein–protein interactions, yielding a modular and programmable system for strong transcriptional activation of multiple orthogonal synthetic transcription factor variants in Escherichia coli. To our knowledge this is the first exogenous, programmable activator system in bacteria.

show abstract

Section: Introductionmentioning

confidence: 99%

Programmable T7-based synthetic transcription factors

Hussey

McMillen

2018

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…Such dual selections have been accomplished by sorting cells based low and high fluorescence, utilizing proteins that can be both toxic and selective ( e.g. , TetA or HSV-TK), or by deploying separate positive and negative selections 26, 35, 37-44 . This has been applied to improving the response functions and eliminating cross-reactivity between pairs of regulators 23, 26, 35, 45 .…”

mentioning

confidence: 99%

Marionette:E. colicontaining 12 highly-optimized small molecule sensors

Meyer

Segall-Shapiro

2018

Preprint

View full text Add to dashboard Cite

2 Cellular processes are carried out by many interacting genes and their study and optimization requires 29 multiple levers by which they can be independently controlled. The most common method is via a 30 genetically-encoded sensor that responds to a small molecule (an "inducible system"). However, these 31 sensors are often suboptimal, exhibiting high background expression and low dynamic range. Further, 32 using multiple sensors in one cell is limited by cross-talk and the taxing of cellular resources. Here, we 33 have developed a directed evolution strategy to simultaneously select for less background, high 34 dynamic range, increased sensitivity, and low crosstalk. Libraries of the regulatory protein and output 35 promoter are built based on random and rationally-guided mutations. This is applied to generate a set 36 of 12 high-performance sensors, which exhibit >100-fold induction with low background and cross-37 reactivity. These are combined to build a single "sensor array" and inserted into the genomes of E. coli 38 MG1655 (wild-type), DH10B (cloning), and BL21 (protein expression). These "Marionette" strains allow 39for the independent control of gene expression using 2,4-diacetylphophloroglucinol (DAPG), cuminic 40 Advances in biology are often tied to new methods that use external stimuli to control the levels 45 of gene expression [1][2][3] . Pioneered in the early 1980s, so-called inducible systems were developed that allow 46 genes to be turned on by adding a small molecule inducer to the growth media 4 . These consist of a protein 47 transcription factor (e.g., LacI) whose binding to a DNA operator in a promoter is controlled by the inducer 48 (e.g., IPTG). Initially co-opted from natural regulatory networks, over the years many versions were 49 designed to improve performance. In the 1990s, additional systems were developed that responded to 50 other inducers, notably arabinose and aTc, which became common tools in the field. In 1997, Lutz and 51Bujard published a seminal paper that combined three (IPTG, arabinose, aTc) that could be easily 52 interchanged on a two-plasmid system 5 . Its organizational simplicity, compatibility, and quantified 53 response functions were revolutionary. Beyond providing a new tool to biologists to control multiple 54 genes with independent "strings," it facilitated researchers with quantitative backgrounds to enter 55 biology [6][7] . Armed with the new ability to control two genes with precision, physicists and engineers built 56 the first synthetic genetic circuits, performed single molecule experiments inside cells, deconstructed the 57 origins of noise in gene expression, determined how enzyme balancing impacts metabolic flux, elucidated 58 rules underlying the assembly of molecular machines, and built synthetic symbiotic microbial 59 communities, just to highlight a few [8][9][10][11][12][13][14][15][16][17][18][19] . 60. 20, 2018; 3 Sensor performance is quantified by its response function; in other words, how the concentration 61 of inducer changes th...

show abstract

“…35 Consequently, the bacteriophage T7 RNA polymerase has been designed for various purposes. [36][37][38] TF, with a high dynamic response range and sensitivity to inducers, is highly desirable for genetic engineering and synthetic biology. Hou et al constructed ultrasensitive TFs by including oligomerizing proteins to enable more stringent control via the formation of a loop structure of the promoter region.…”

Section: Employing Compatible Heterogeneous Tfsmentioning

confidence: 99%

Transcriptional factor engineering in microbes for industrial biotechnology

Wang

Liang

Xing

et al. 2020

J of Chemical Tech & Biotech

View full text Add to dashboard Cite

Transcription can be regulated by controlling the provision of transcriptional factors (TFs), because TFs determine the transcription process by specifying the binding of RNA polymerase holoenzyme on promoters. Manipulations, such as replacing endogenous TFs with mutants obtained through directed evolution (or rational design), and introducing compatible heterogeneous TFs, are effective ways to regulate transcription. Designing artificial TFs with desired properties by following the principles of protein design and reconstructing ancestral TFs with the information gained from a perspective of evolution by multiple sequence alignments of the related TFs, are feasible alternative options. The engineered TFs can be used to create inducible protein expression systems that respond to a specific stimulus. Moreover, the engineered TFs could lead to a transcriptional profile different from that of the wild-type strains. Accordingly, these engineered TFs could be utilized to construct strains resistant to adverse conditions, since the emergence of resistance generally requires global transcriptional changes at the transcriptomic scale. Meanwhile, these engineered TFs can also be employed in the construction of sophisticatedly designed genetic circuits for diverse purposes. In this paper, we reviewed the advances in the above-mentioned aspects.

show abstract

Evolutionary Design of Choline-Inducible and -Repressible T7-Based Induction Systems

Cited by 24 publications

References 65 publications

Programmable T7-based synthetic transcription factors

Programmable T7-based synthetic transcription factors

Marionette:E. colicontaining 12 highly-optimized small molecule sensors

Transcriptional factor engineering in microbes for industrial biotechnology

Contact Info

Product

Resources

About