Orthogonality and Burdens of Heterologous AND Gate Gene Circuits in <i>E. coli</i>

Synthetic biology approaches commonly introduce heterologous gene networks into a host to predictably program cells, with the expectation of the synthetic network being orthogonal to the host background. However, introduced circuits may interfere with the host’s physiology, either indirectly by posing a metabolic burden and/or through unintended direct interactions between parts of the circuit with those of the host, affecting functionality. Here we used RNA-Seq transcriptome analysis to quantify the interactions between a representative heterologous AND gate circuit and the host Escherichia coli under various conditions including circuit designs and plasmid copy numbers. We show that the circuit plasmid copy number outweighs circuit composition for their effect on host gene expression with medium-copy number plasmid showing more prominent interference than its low-copy number counterpart. In contrast, the circuits have a stronger influence on the host growth with a metabolic load increasing with the copy number of the circuits. Notably, we show that variation of copy number, an increase from low to medium copy, caused different types of change observed in the behaviour of components in the AND gate circuit leading to the unbalance of the two gate-inputs and thus counterintuitive output attenuation. The study demonstrates the circuit plasmid copy number is a key factor that can dramatically affect the orthogonality, burden and functionality of the heterologous circuits in the host chassis. The results provide important guide for future efforts to design orthogonal and robust gene circuits with minimal unwanted interaction and burden to their host

Engineering the Ultrasensitive Transcription Factors by Fusing a Modular Oligomerization Domain

The dimerization and high-order oligomerization of transcription factors has endowed them with cooperative regulatory capabilities that play important roles in many cellular functions. However, such advanced regulatory capabilities have not been fully exploited in synthetic biology and genetic engineering. Here, we engineered a C-terminally fused oligomerization domain to improve the cooperativity of transcription factors. First, we found that two of three designed oligomerization domains significantly increased the cooperativity and ultrasensitivity of a transcription factor for the regulated promoter. Then, seven additional transcription factors were used to assess the modularity of the oligomerization domains, and their ultrasensitivity was generally improved, as assessed by their Hill coefficients. Moreover, we also demonstrated that the allosteric capability of the ligand-responsive domain remained intact when fusing with the designed oligomerization domain. As an example application, we showed that the engineered ultrasensitive transcription factor could be used to significantly improve the performance of a “stripe-forming” gene circuit. We envision that the oligomerization modules engineered in this study could act as a powerful tool to rapidly tune the underlying response profiles of synthetic gene circuits and metabolic pathway controllers

Ribozyme-based insulator parts buffer synthetic circuits from genetic context

Synthetic genetic programs are built from circuits that integrate sensors and implement temporal control of gene expression. Transcriptional circuits are layered by using promoters to carry the signal between circuits. In other words, the output promoter of one circuit serves as the input promoter to the next. Thus, connecting circuits requires physically connecting a promoter to the next circuit. We show that the sequence at the junction between the input promoter and circuit can affect the input-output response (transfer function) of the circuit. A library of putative sequences that might reduce (or buffer) such context effects, which we refer to as 'insulator parts', is screened in Escherichia coli. We find that ribozymes that cleave the 5′ untranslated region (5′-UTR) of the mRNA are effective insulators. They generate quantitatively identical transfer functions, irrespective of the identity of the input promoter. When these insulators are used to join synthetic gene circuits, the behavior of layered circuits can be predicted using a mathematical model. The inclusion of insulators will be critical in reliably permuting circuits to build different programs.Life Technologies, Inc.United States. Defense Advanced Research Projects Agency (DARPA CLIO N66001-12-C-4018)United States. Office of Naval Research (N00014-10-1-0245)National Science Foundation (U.S.) (CCF-0943385)National Institutes of Health (U.S.) (AI067699)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SynBERC, SA5284-11210

Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit

Abstract Background Plasmid-borne genetic editing tools, including the widely used CRISPR-Cas9 system, have greatly facilitated bacterial programming to obtain novel functionalities. However, the lack of effective post-editing plasmid elimination methods impedes follow-up genetic manipulation or application. Conventional strategies including exposure to physical and chemical treatments, or exploiting temperature-sensitive replication origins have several drawbacks (e.g., they are limited for efficiency and are time-consuming). Therefore, the demand is apparent for easy and rapid elimination of the tool plasmids from their bacterial hosts after genetic manipulation. Results To bridge this gap, we designed a novel EXIT circuit with the homing endonuclease, which can be exploited for rapid and efficient elimination of various plasmids with diverse replication origins. As a proof of concept, we validated the EXIT circuit in Escherichia coli by harnessing homing endonuclease I-SceI and its cleavage site. When integrated into multiple plasmids with different origins, the EXIT circuit allowed them to be eliminated from the host cells, simultaneously. By combining the widely used plasmid-borne CRISPR-Cas9 system and the EXIT circuit, we constructed an easy-to-use CRISPR-Cas9 system that eliminated the Cas9- and the single-guide RNA (sgRNA)-encoding plasmids in one-step. Within 3 days, we successfully constructed an atrazine-degrading E. coli strain, thus further demonstrating the advantage of this new CRISPR-Cas9 system for bacterial genome editing. Conclusions Our novel EXIT circuit, which exploits the homing endonuclease I-SceI, enables plasmid(s) with different replication origins to be eliminated from their host cells rapidly and efficiently. We also developed an easy-to-use CRISPR-Cas9 system with the EXIT circuit, and this new system can be widely applied to bacterial genome editing

A flexible, modular and versatile functional part assembly toolkit for gene cluster engineering in Streptomyces

Streptomyces has enormous potential to produce novel natural products (NPs) as it harbors a huge reservoir of uncharacterized and silent natural product biosynthetic gene clusters (BGCs). However, the lack of efficient gene cluster engineering strategies has hampered the pace of new drug discovery. Here, we developed an easy-to-use, highly flexible DNA assembly toolkit for gene cluster engineering. The DNA assembly toolkit is compatible with various DNA assembling approaches including Biobrick, Golden Gate, CATCH, yeast homologous recombination-based DNA assembly and homing endonuclease-mediated assembly. This compatibility offers great flexibility in handling multiple genetic parts or refactoring large gene clusters. To demonstrate the utility of this toolkit, we quantified a library of modular regulatory parts, and engineered a gene cluster (act) using characterized promoters that led to increased production. Overall, this work provides a powerful part assembly toolkit that can be used for natural product discovery and optimization in Streptomyces

Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous Streptomyces Hosts

Thaxtomin A is a potent bioherbicide in both organic and conventional agriculture; however, its low yield hinders its wide application. Here, we report the direct cloning and heterologous expression of the thaxtomin A gene cluster in three well-characterized Streptomyces hosts. Then, we present an efficient, markerless and multiplex large gene cluster editing method based on in vitro CRISPR/Cas9 digestion and yeast homologous recombination. With this method, we successfully engineered the thaxtomin A cluster by simultaneously replacing the native promoters of the txtED operon, txtABH operon and txtC gene with strong constitutive promoters, and the yield of thaxtomin A improved to 289.5 &micro;g/mL in heterologous Streptomyces coelicolor M1154. To further optimize the biosynthetic pathway, we used constraint-based combinatorial design to build 27 refactored gene clusters by varying the promoter strength of every operon, and the highest titer of thaxtomin A production reached 504.6 &mu;g/mL. Taken altogether, this work puts forward a multiplexed promoter engineering strategy to engineer secondary metabolism gene clusters for efficiently improving fermentation titers

Engineered DNase-inactive Cpf1 variants to improve targeting scope for base editing in E. coli

The development of base editing (BE) technology has opened a new avenue for research studies in bacteriology, particularly for bacterial species in which the DNA double-strand breaks (DSBs) introduced by CRISPR/Cas system would lead to cell death. However, a major limitation of BE-mediated gene editing is the restricted editable sites in the target bacterial genome due to highly diverse genomic compositions, such as GC content. Herein, we developed a broad-spectrum DNase-inactive Cpf1 (dCpf1) variant from Francisella novicida (bsdFnCpf1) through directed evolution. The resulting optimized mutant showed a substantially expanded targeting range, including previously non-canonical protospacer-adjacent motifs (PAMs), especially the GC-rich PAMs. Cytidine deaminase APOBEC1 and uracil DNA glycosylase inhibitor (UGI) were fused with bsdFnCpf1 to achieve specific C to T mutations at multiple target sites with canonical or non-canonical PAMs in the E. coli genome without compromising cell growth. We anticipate that bsdFnCpf1 could be applied for multiplex gene regulation and BE in species that have been reported to be suitable for Cpf1

Improving cooperativity of transcription activators by oligomerization domains in mammalian cells

Cooperative activation is critical for the applications of synthetic biology in mammalian cells. In this study, we have developed cooperative transcription factor by fusing oligomerization domain in mammalian cells. Firstly, we demonstrated that two oligomerized domains (CI434 and CI) successfully improved transcription factor cooperativity in bacterial cells but failed to increase cooperativity in mammalian cells, possibly because the additional mammalian activation domain disrupted their oligomerization capability. Therefore, we chose a different type of oligomerized domain (CarHC), whose ability to oligomerize is not dependent on its C-terminal domains, to fuse with a transcription factor (RpaR) and activation domain (VTR3), forming a potential cooperative transcription activator RpaR-CarH-VTR3 for mammalian regulatory systems. Compared with RpaR-VTR3, the cooperativity of RpaR-CarH-VTR3 was significantly improved with higher Hill coefficient and a narrower input range in the inducible switch system in mammalian cells. Moreover, a mathematical model based on statistical mechanics model was developed and the simulation results supported the hypothesis that the tetramer of the CarH domain in mammalian cells was the reason for the cooperative capacity of RpaR-CarH-VTR3

The Topological Characteristics of Biological Ratio-Sensing Networks

Ratio sensing is a fundamental biological function observed in signal transduction and decision making. In the synthetic biology context, ratio sensing presents one of the elementary functions for cellular multi-signal computation. To investigate the mechanism of the ratio-sensing behavior, we explored the topological characteristics of biological ratio-sensing networks. With exhaustive enumeration of three-node enzymatic and transcriptional regulatory networks, we found that robust ratio sensing was highly dependent on network structure rather than network complexity. Specifically, a set of seven minimal core topological structures and four motifs were deduced to be capable of robust ratio sensing. Further investigations on the evolutionary space of robust ratio-sensing networks revealed highly clustered domains surrounding the core motifs which suggested their evolutionary plausibility. Our study revealed the network topological design principles of ratio-sensing behavior and provided a design scheme for constructing regulatory circuits with ratio-sensing behavior in synthetic biology