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Rationally designed bidirectional promoter improves the evolutionary stability of synthetic genetic circuits.

ABSTRACT: One problem with synthetic genes in genetically engineered organisms is that these foreign DNAs will eventually lose their functions over evolutionary time in absence of selective pressures. This general limitation can restrain the long-term study and industrial application of synthetic genetic circuits. Previous studies have shown that because of their crucial regulatory functions, prokaryotic promoters in synthetic genetic circuits are especially vulnerable to mutations. In this study, we rationally designed robust bidirectional promoters (BDPs), which are self-protected through the complementarity of their overlapping forward and backward promoter sequences on DNA duplex. When the transcription of a target non-essential gene (e.g. green fluorescent protein) was coupled to the transcription of an essential gene (e.g. antibiotic resistance gene) through the BDP, the evolutionary half-time of the gene of interest increases 4-10 times, depending on the strain and experimental conditions used. This design of using BDPs to increase the mutational stability of genetic circuits can be potentially applied to synthetic biology applications in general.

SUBMITTER: Yang S

PROVIDER: S-EPMC3592475 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Rationally designed bidirectional promoter improves the evolutionary stability of synthetic genetic circuits.

Yang Song S Sleight Sean C SC Sauro Herbert M HM

Nucleic acids research 20121023 1

One problem with synthetic genes in genetically engineered organisms is that these foreign DNAs will eventually lose their functions over evolutionary time in absence of selective pressures. This general limitation can restrain the long-term study and industrial application of synthetic genetic circuits. Previous studies have shown that because of their crucial regulatory functions, prokaryotic promoters in synthetic genetic circuits are especially vulnerable to mutations. In this study, we rati ...[more]

PMID: 23093602

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Project description:BackgroundThe oleaginous yeast Yarrowia lipolytica is increasingly used as a chassis strain for generating bioproducts. Several hybrid promoters with different strengths have been developed by combining multiple copies of an upstream activating sequence (UAS) associated with a TATA box and a core promoter. These promoters display either constitutive, phase-dependent, or inducible strong expression. However, there remains a lack of bidirectional inducible promoters for co-expressing genes in Y. lipolytica.ResultsThis study built on our previous work isolating and characterizing the UAS of the erythritol-induced genes EYK1 and EYD1 (UAS-eyk1). We found an erythritol-inducible bidirectional promoter (BDP) located in the EYK1-EYL1 intergenic region. We used the BDP to co-produce YFP and RedStarII fluorescent proteins and demonstrated that the promoter's strength was 2.7 to 3.5-fold stronger in the EYL1 orientation compared to the EYK1 orientation. We developed a hybrid erythritol-inducible bidirectional promoter (HBDP) containing five copies of UAS-eyk1 in both orientations. It led to expression levels 8.6 to 19.2-fold higher than the native bidirectional promoter. While the BDP had a twofold-lower expression level than the strong constitutive TEF promoter, the HBDP had a 5.0-fold higher expression level when oriented toward EYL1 and a 2.4-fold higher expression level when oriented toward EYK1. We identified the optimal media for BDP usage by exploring yeast growth under microbioreactor conditions. Additionally, we constructed novel Golden Gate biobricks and a destination vector for general use.ConclusionsIn this research, we developed novel bidirectional and hybrid bidirectional promoters of which expression can be fine-tuned, responding to the need for versatile promoters in the yeast Y. lipolytica. This study provides effective tools that can be employed to smoothly adjust the erythritol-inducible co-expression of two target genes in biotechnology applications. BDPs developed in this study have potential applications in the fields of heterologous protein production, metabolic engineering, and synthetic biology.

Project description:BackgroundOne problem with engineered genetic circuits in synthetic microbes is their stability over evolutionary time in the absence of selective pressure. Since design of a selective environment for maintaining function of a circuit will be unique to every circuit, general design principles are needed for engineering evolutionary robust circuits that permit the long-term study or applied use of synthetic circuits.ResultsWe first measured the stability of two BioBrick-assembled genetic circuits propagated in Escherichia coli over multiple generations and the mutations that caused their loss-of-function. The first circuit, T9002, loses function in less than 20 generations and the mutation that repeatedly causes its loss-of-function is a deletion between two homologous transcriptional terminators. To measure the effect between transcriptional terminator homology levels and evolutionary stability, we re-engineered six versions of T9002 with a different transcriptional terminator at the end of the circuit. When there is no homology between terminators, the evolutionary half-life of this circuit is significantly improved over 2-fold and is independent of the expression level. Removing homology between terminators and decreasing expression level 4-fold increases the evolutionary half-life over 17-fold. The second circuit, I7101, loses function in less than 50 generations due to a deletion between repeated operator sequences in the promoter. This circuit was re-engineered with different promoters from a promoter library and using a kanamycin resistance gene (kanR) within the circuit to put a selective pressure on the promoter. The evolutionary stability dynamics and loss-of-function mutations in all these circuits are described. We also found that on average, evolutionary half-life exponentially decreases with increasing expression levels.ConclusionsA wide variety of loss-of-function mutations are observed in BioBrick-assembled genetic circuits including point mutations, small insertions and deletions, large deletions, and insertion sequence (IS) element insertions that often occur in the scar sequence between parts. Promoter mutations are selected for more than any other biological part. Genetic circuits can be re-engineered to be more evolutionary robust with a few simple design principles: high expression of genetic circuits comes with the cost of low evolutionary stability, avoid repeated sequences, and the use of inducible promoters increases stability. Inclusion of an antibiotic resistance gene within the circuit does not ensure evolutionary stability.

Project description:For practical construction of complex synthetic genetic networks able to perform elaborate functions it is important to have a pool of relatively simple modules with different functionality which can be compounded together. To complement engineering of very different existing synthetic genetic devices such as switches, oscillators or logical gates, we propose and develop here a design of synthetic multi-input classifier based on a recently introduced distributed classifier concept. A heterogeneous population of cells acts as a single classifier, whose output is obtained by summarizing the outputs of individual cells. The learning ability is achieved by pruning the population, instead of tuning parameters of an individual cell. The present paper is focused on evaluating two possible schemes of multi-input gene classifier circuits. We demonstrate their suitability for implementing a multi-input distributed classifier capable of separating data which are inseparable for single-input classifiers, and characterize performance of the classifiers by analytical and numerical results. The simpler scheme implements a linear classifier in a single cell and is targeted at separable classification problems with simple class borders. A hard learning strategy is used to train a distributed classifier by removing from the population any cell answering incorrectly to at least one training example. The other scheme implements a circuit with a bell-shaped response in a single cell to allow potentially arbitrary shape of the classification border in the input space of a distributed classifier. Inseparable classification problems are addressed using soft learning strategy, characterized by probabilistic decision to keep or discard a cell at each training iteration. We expect that our classifier design contributes to the development of robust and predictable synthetic biosensors, which have the potential to affect applications in a lot of fields, including that of medicine and industry.

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Rationally designed bidirectional promoter improves the evolutionary stability of synthetic genetic circuits.

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Rationally designed bidirectional promoter improves the evolutionary stability of synthetic genetic circuits.

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