State of the Art of Genetic Engineering in Potato: From the First Report to Its Future Potential

Potato (Solanum tuberosum L.) is a crop of world importance that produces tubers of high nutritional quality. It is considered one of the promising crops to overcome the challenges of poverty and hunger worldwide. However, it is exposed to different biotic and abiotic stresses that can cause significant losses in production. Thus, potato is a candidate of special relevance for improvements through conventional breeding and biotechnology. Since conventional breeding is time-consuming and challenging, genetic engineering provides the opportunity to introduce/switch-off genes of interest without altering the allelic combination that characterize successful commercial cultivars or to induce targeted sequence modifications by New Breeding Techniques. There is a variety of methods for potato improvement via genetic transformation. Most of them incorporate genes of interest into the nuclear genome; nevertheless, the development of plastid transformation protocols broadened the available approaches for potato breeding. Although all methods have their advantages and disadvantages, Agrobacterium-mediated transformation is the most used approach. Alternative methods such as particle bombardment, protoplast transfection with polyethylene glycol and microinjection are also effective. Independently of the DNA delivery approach, critical steps for a successful transformation are a rapid and efficient regeneration protocol and a selection system. Several critical factors affect the transformation efficiency: vector type, insert size, Agrobacterium strain, explant type, composition of the subculture media, selective agent, among others. Moreover, transient or stable transformation, constitutive or inducible promoters, antibiotic/herbicide resistance or marker-free strategies can be considered. Although great efforts have been made to optimize all the parameters, potato transformation protocols are highly genotype-dependent. Genome editing technologies provide promising tools in genetic engineering allowing precise modification of targeted sequences. Interestingly, transient expression of genome editing components in potato protoplasts was reported to generate edited plants without the integration of any foreign DNA, which is a valuable aspect from both a scientific and a regulatory perspective. In this review, current challenges and opportunities concerning potato genetic engineering strategies developed to date are discussed. We describe their critical parameters and constrains, and the potential application of the available tools for functional analyses or biotechnological purposes. Public concerns and safety issues are also addressed.

Independent control of mean and noise by convolution of gene expression distributions

Gene expression noise can reduce cellular fitness or facilitate processes such as alternative metabolism, antibiotic resistance, and differentiation. Unfortunately, efforts to study the impacts of noise have been hampered by a scaling relationship between noise and expression level from individual promoters. Here, we use theory to demonstrate that mean and noise can be controlled independently by expressing two copies of a gene from separate inducible promoters in the same cell. We engineer low and high noise inducible promoters to validate this result in Escherichia coli, and develop a model that predicts the experimental distributions. Finally, we use our method to reveal that the response of a promoter to a repressor is less sensitive with higher repressor noise and explain this result using a law from probability theory. Our approach can be applied to investigate the effects of noise on diverse biological pathways or program cellular heterogeneity for synthetic biology applications.

Poplar Allene Oxide Synthase 1 Gene Promoter Drives Rapid and Localized Expression by Wounding

Promoters play critical roles in controlling the transcription of genes and are important as tools to drive heterologous expression for biotechnological applications. In addition to core transcription factor-binding motifs that assist in the binding of RNA polymerases, there are specific nucleotide sequences in a promoter region to allow regulation of gene expression. The allene oxide synthase (AOS) gene family are cytochrome P450s that are responsive to a variety of environmental stress, making them good candidates for the discovery of inducible promoters. Populus AOS homologs separate phylogenetically into two clades. Based on the 19 promoter motifs with significant abundance differences between the two clades, Clade I AOS genes are likely more responsive to hormones, salt, and pathogen, whereas clade II homologs are likely inducible by water stress. In this study, an upstream promoter from a Clade I poplar AOS encoding gene (AOS1) was cloned and used to drive the expression of a ß-glucuronidase (GUS) gene in Arabidopsis. AOS is an essential enzyme in the lipoxygenase pathway that is responsible for the production of many non-volatile oxylipins in plants, including the jasmonates, which are regulatory phytohormones coordinating a variety of biological and stress response functions. Consistent with AOS transcript expression patterns, we found that the poplar AOS1 promoter drives rapid and localized expression by wounding. The study provides insight on the responsive elements in the poplar AOS promoters, but more importantly identifies a strong wound-inducible and localized promoter for future applications.

PccGEO: prior constraints conditioned genetic elements optimization

Functional genetic elements are one of the most essential units for synthetic biology. However, both knowledge-driven and data-driven methodology can hardly accomplish the complicated task of genetic elements design efficiently due to the lack of explicit regulatory logics and training samples. Here, we proposed a knowledge-constraint deep learning model named PccGEO to automatically design functional genetic elements with high success rate and efficiency. PccGEO utilized a novel "fill-in-the-flank" strategy with a conditional generative adversarial network structure to optimize the flanking regions of known functional sequences derived from the biological prior knowledge, which can efficiently capture the implicit patterns with a reduced searching space. We applied PccGEO in the design of Escherichia coli promoters, and found that the implicit patterns in flanking regions matter to the properties of promoters such as the expression level. The PccGEO-designed constitutive and inducible promoters showed more than 91.6% chance of success by in vivo validation. We further utilized PccGEO by setting a limited frequency of nucleotide modifications and surprisingly found that the expression level of E. coli sigma 70 promoters could show up to a 159.3-fold increase with only 10-bp nucleotide modifications. The results supported that the implicit patterns are important in the design of functional gene elements and validated the strong capacity of our method in the efficient design of functional genetic elements.

Transcriptional interference in toehold switch-based RNA circuits

Gene regulation based on regulatory RNA is an important mechanism in cells and is increasingly used for regulatory circuits in synthetic biology. Toehold switches are rationally designed post-transcriptional riboregulators placed in the 5' untranslated region of mRNA molecules. In the inactive state of a toehold switch, the ribosome-binding site is inaccessible for the ribosome. In the presence of a trigger RNA molecule protein production is turned on. Using antisense RNA against trigger molecules (anti-trigger RNA), gene expression can also be switched off again. We here study the utility and regulatory effect of antisense transcription in this context, which enables a particularly compact circuit design. Our circuits utilize two inducible promoters that separately regulate trigger and anti-trigger transcription, whereas their cognate toehold switch, regulating expression of a reporter protein, is transcribed from a constitutive promoter. We explore various design options for the arrangement of the promoters and demonstrate that the resulting dynamic behavior is strongly influenced by transcriptional interference (TI) effects, leading to more than four-fold differences in expression levels. Our experimental results are consistent with previous findings that enhanced local RNA polymerase concentrations due to active promoters in close proximity lead to an increase in transcriptional activity of the strongest promoter in the circuits. Based on this insight, we selected optimum promoter designs and arrangements for the realization of a genetic circuit comprised of two toehold switches, two triggers and two anti-triggers that function as a post-transcriptional RNA regulatory exclusive OR (XOR) gate.

Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9

Despite four decades of research to support the association between DNA methylation and gene expression, the causality of this relationship remains unresolved. Here, we reaffirm that experimental confounds preclude resolution of this question with existing strategies, including recently developed CRISPR/dCas9 and TET-based epigenetic editors. Instead, we demonstrate a highly effective method using only nuclease-dead Cas9 and guide RNA to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzyme, thereby enabling the examination of the role of DNA demethylation per se in living cells, with no evidence of off-target activity. Using this method, we probe a small number of inducible promoters and find the effect of DNA demethylation to be small, while demethylation of CpG-rich FMR1 produces larger changes in gene expression. This method could be used to reveal the extent and nature of the contribution of DNA methylation to gene regulation.

Construction of multicellular yeast networks using the communication toolkit with variable specificity and attenuation

The key next step in synthetic biology is to extend cellular network engineering to the multicellular level by utilizing cell-cell communication for information processing. To facilitate the implementation of multicellular networks in the most commonly used eukaryotic chassis, Saccharomyces cerevisiae, we developed the yeast communication toolkit (YCTK). This toolkit is based on the fungal mating pathway and contains five pheromone-inducible promoters (response parts), eleven pheromones (α-factors; sender parts), eleven pheromone receptors (Ste2; receiver parts), as well as five Bar1 proteases (suppressor parts). All YCTK parts were thoroughly characterized and are compatible with the commonly used yeast Golden Gate cloning standard. We demonstrated the application of the YCTK by implementing several different logic gate-like population networks. Furthermore, we used this toolkit to investigate the pheromone-receptor promiscuity patterns among different yeast species. This toolkit extends currently available resources for construction of complex multicellular eukaryotic networks with varying degrees of promiscuity and attenuation.

Constructing of Bacillus subtilis-Based Lux-Biosensors with the Use of Stress-Inducible Promoters

Here, we present a new lux-biosensor based on Bacillus subtilis for detecting of DNA-tropic and oxidative stress-causing agents. Hybrid plasmids pNK-DinC, pNK-AlkA, and pNK-MrgA have been constructed, in which the Photorhabdus luminescens reporter genes luxABCDE are transcribed from the stress-inducible promoters of B. subtilis: the SOS promoter PdinC, the methylation-specific response promoter PalkA, and the oxidative stress promoter PmrgA. The luminescence of B. subtilis-based biosensors specifically increases in response to the appearance in the environment of such common toxicants as mitomycin C, methyl methanesulfonate, and H2O2. Comparison with Escherichia coli-based lux-biosensors, where the promoters PdinI, PalkA, and Pdps were used, showed generally similar characteristics. However, for B. subtilis PdinC, a higher response amplitude was observed, and for B. subtilis PalkA, on the contrary, both the amplitude and the range of detectable toxicant concentrations were decreased. B. subtilis PdinC and B. subtilis PmrgA showed increased sensitivity to the genotoxic effects of the 2,2′-bis(bicyclo [2.2.1] heptane) compound, which is a promising propellant, compared to E. coli-based lux-biosensors. The obtained biosensors are applicable for detection of toxicants introduced into soil. Such bacillary biosensors can be used to study the differences in the mechanisms of toxicity against Gram-positive and Gram-negative bacteria.

Induzierbare Promotoren für Cyanobakterien

Cyanobacteria are versatile organisms with extreme phylogenetic diversity. With increasing need for alternative sustainable solutions, they are becoming increasingly attractive as production hosts in the biotechnology sector. Opposed to established heterotrophic model organisms, newer production hosts still require extensive characterization of standard biological parts in order to build complex networks and circuits. Here, we present examples of quantitative characterization of inducible promoters in the model organism Synechocystis sp. PCC 6803.

Optimization of phosphate-limited autoinduction broth for two-stage heterologous protein expression in Escherichia coli

Autoinducible, two-stage protein expression leveraging phosphate-inducible promoters has been recently shown to enable not only high protein titers but also consistent performance across scales from screening systems (microtiter plates) to instrumented bioreactors. However, to date, small-scale production using microtiter plates and shake flasks relies on a complex autoinduction broth (AB) that requires making numerous media components, not all amenable to autoclaving. In this report, the authors develop a simpler media formulation (AB-2) with just a few autoclavable components. AB-2 is robust to small changes in its composition and performs equally, if not better, than AB across different scales. AB-2 will facilitate the adoption of phosphate-limited two-stage protein expression protocols.