2022
Matthew Tridgett; Maria Ababi; Alfonso Jaramillo
Lambda red recombineering of bacteriophage in the lysogenic state Journal Article
In: Methods in molecular biology, vol. 2479, pp. (in press), 2022, ISBN: 9781071622322.
@article{tridgett_lambda_2022,
title = {Lambda red recombineering of bacteriophage in the lysogenic state},
author = {Matthew Tridgett and Maria Ababi and Alfonso Jaramillo},
url = {https://link.springer.com/book/9781071622339},
isbn = {9781071622322},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Methods in molecular biology},
volume = {2479},
pages = {(in press)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Maria Ababi; Matthew Tridgett; Alexander Osgerby; Alfonso Jaramillo
Scarless ssDNA Recombineering of Phage in the Lysogenic State Journal Article
In: Methods in molecular biology (Clifton, N.J.), vol. 2479, pp. (in press), 2022, ISBN: 9781071622322.
@article{ababi_scarless_2022,
title = {Scarless ssDNA Recombineering of Phage in the Lysogenic State},
author = {Maria Ababi and Matthew Tridgett and Alexander Osgerby and Alfonso Jaramillo},
url = {https://link.springer.com/book/9781071622339},
isbn = {9781071622322},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
pages = {(in press)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2021
Daniel Camsund; Alfonso Jaramillo*; Peter Lindblad*
Engineering of a Promoter Repressed by a Light-Regulated Transcription Factor in Escherichia coli Journal Article
In: BioDesign Research, 2021.
Links | BibTeX | Altmetric | Tags:
@article{Alfonso_Jaramillo_100687242,
title = {Engineering of a Promoter Repressed by a Light-Regulated Transcription Factor in Escherichia coli},
author = {Daniel Camsund and Alfonso Jaramillo* and Peter Lindblad*},
url = {http://doi.org/10.34133/2021/9857418},
doi = {10.34133/2021/9857418},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {BioDesign Research},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Satya Prakash; Adrian Racovita; Clenira Varela; Mark Walsh; Roberto Galizi; Mark Isalan; Alfonso Jaramillo
Engineering Adaptive Gene Circuits in Bacteria Mastering Game Playing by Reinforcement Learning Journal Article
In: Biophysical Journal, 2021.
Links | BibTeX | Altmetric | Tags:
@article{Alfonso_Jaramillo_102297231,
title = {Engineering Adaptive Gene Circuits in Bacteria Mastering Game Playing by Reinforcement Learning},
author = {Satya Prakash and Adrian Racovita and Clenira Varela and Mark Walsh and Roberto Galizi and Mark Isalan and Alfonso Jaramillo},
url = {http://doi.org/10.1016/J.BPJ.2020.11.1683},
doi = {10.1016/J.BPJ.2020.11.1683},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Biophysical Journal},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2020
Roberto Galizi; John N. Duncan; William Rostain; Charlotte M Quinn; Marko Storch; Manish Kushwaha; Alfonso Jaramillo
Engineered RNA-Interacting CRISPR Guide RNAs for Genetic Sensing and Diagnostics Journal Article
In: The CRISPR Journal, vol. 3, no. 5, pp. 398–408, 2020.
Links | BibTeX | Altmetric | Tags:
@article{Galizi_2020,
title = {Engineered RNA-Interacting CRISPR Guide RNAs for Genetic Sensing and Diagnostics},
author = {Roberto Galizi and John N. Duncan and William Rostain and Charlotte M Quinn and Marko Storch and Manish Kushwaha and Alfonso Jaramillo},
url = {https://doi.org/10.1089%2Fcrispr.2020.0029},
doi = {10.1089/crispr.2020.0029},
year = {2020},
date = {2020-10-01},
urldate = {2020-10-01},
journal = {The CRISPR Journal},
volume = {3},
number = {5},
pages = {398--408},
publisher = {Mary Ann Liebert Inc},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Andreas K. Broedel; Rui Rodrigues; Alfonso Jaramillo; Mark Isalan
Accelerated evolution of a minimal 63-amino acid dual transcription factor Journal Article
In: Science advances, vol. 6, no. 24, pp. eaba2728, 2020, (2 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{broedel_accelerated_2020,
title = {Accelerated evolution of a minimal 63-amino acid dual transcription factor},
author = {Andreas K. Broedel and Rui Rodrigues and Alfonso Jaramillo and Mark Isalan},
doi = {10.1126/sciadv.aba2728},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Science advances},
volume = {6},
number = {24},
pages = {eaba2728},
abstract = {Transcription factors control gene expression in all life. This raises the question of what is the smallest protein that can support such activity. In nature, Cro from bacteriophage λ is one of the smallest known repressors (66 amino acids), and activators are typically much larger (e.g., λ cI, 237 amino acids). Previous efforts to engineer a minimal activator from λ Cro resulted in no activity in vivo in cells. In this study, we show that directed evolution results in a new Cro activator-repressor that functions as efficiently as λ cI in vivo. To achieve this, we develop phagemid-assisted continuous evolution (PACEmid). We find that a peptide as small as 63 amino acids functions efficiently as an activator and/or repressor. To our knowledge, this is the smallest protein activator that enables polymerase recruitment, highlighting the capacity of transcription factors to evolve from very short peptide sequences.},
note = {2 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Aurelija M. Grigonyte; Christian Harrison; Paul R. MacDonald; Ariadna Montero-Blay; Matthew Tridgett; John Duncan; Antonia P. Sagona; Chrystala Constantinidou; Alfonso Jaramillo*; Andrew Millard*
Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7 Journal Article
In: Viruses, vol. 12, no. 2, pp. 193, 2020, (5 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{grigonyte_comparison_2020,
title = {Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7},
author = {Aurelija M. Grigonyte and Christian Harrison and Paul R. MacDonald and Ariadna Montero-Blay and Matthew Tridgett and John Duncan and Antonia P. Sagona and Chrystala Constantinidou and Alfonso Jaramillo* and Andrew Millard*},
doi = {10.3390/v12020193},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Viruses},
volume = {12},
number = {2},
pages = {193},
abstract = {With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes cmk or trxA have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here, we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple T7 tail fibre mutants. Furthermore, we found the type II CRISPR-Cas system is easier to use and generally more efficient than a type I system in the engineering of phage T7. These results provide a foundation for the future, more efficient engineering of bacteriophage T7.},
note = {5 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Matthew Tridgett; Maria Ababi; Alexander Osgerby; Robert Ramirez Garcia; Alfonso Jaramillo
Engineering Bacteria to Produce Pure Phage-like Particles for Gene Delivery Journal Article
In: ACS Synthetic Biology, 2020, (0 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{tridgett_engineering_2020,
title = {Engineering Bacteria to Produce Pure Phage-like Particles for Gene Delivery},
author = {Matthew Tridgett and Maria Ababi and Alexander Osgerby and Robert Ramirez Garcia and Alfonso Jaramillo},
doi = {10.1021/acssynbio.0c00467},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {ACS Synthetic Biology},
abstract = {Natural and engineered phages have been used in many applications, but their use to deliver user-defined genetic cargoes has been hampered by contamination with replicative phage, restricting use of the technology beyond the laboratory. Here we present a method to produce transducing particles without contamination. In addition, we demonstrate the use of a helper phage-free transducing particle preparation as an antimicrobial agent. This will pave the way for the development of new phage-based technologies with greater scope than lytic phage therapy.},
note = {0 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Adrian Racovita; Alfonso Jaramillo
Reinforcement learning in synthetic gene circuits Journal Article
In: Biochemical Society transactions, vol. 48, no. 4, pp. 1637–1643, 2020, (1 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{racovita_reinforcement_2020,
title = {Reinforcement learning in synthetic gene circuits},
author = {Adrian Racovita and Alfonso Jaramillo},
doi = {10.1042/bst20200008},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Biochemical Society transactions},
volume = {48},
number = {4},
pages = {1637--1643},
abstract = {Synthetic gene circuits allow programming in DNA the expression of a phenotype at a given environmental condition. The recent integration of memory systems with gene circuits opens the door to their adaptation to new conditions and their re-programming. This lays the foundation to emulate neuromorphic behaviour and solve complex problems similarly to artificial neural networks. Cellular products such as DNA or proteins can be used to store memory in both digital and analog formats, allowing cells to be turned into living computing devices able to record information regarding their previous states. In particular, synthetic gene circuits with memory can be engineered into living systems to allow their adaptation through reinforcement learning. The development of gene circuits able to adapt through reinforcement learning moves Sciences towards the ambitious goal: the bottom-up creation of a fully fledged living artificial intelligence.},
note = {1 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2019
Xiaohan Yang; Lei S. Qi; Alfonso Jaramillo; Zong-Ming Max Cheng
Biodesign Research to Advance the Principles and Applications of Biosystems Design Journal Article
In: BioDesign Research, vol. 2019, pp. 1–4, 2019, (3 citations (Semantic Scholar/DOI) [2021-05-15]).
Links | BibTeX | Altmetric | Tags:
@article{yang_biodesign_2019,
title = {Biodesign Research to Advance the Principles and Applications of Biosystems Design},
author = {Xiaohan Yang and Lei S. Qi and Alfonso Jaramillo and Zong-Ming Max Cheng},
doi = {10.34133/2019/9680853},
year = {2019},
date = {2019-01-01},
journal = {BioDesign Research},
volume = {2019},
pages = {1--4},
note = {3 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Roberto Galizi; Alfonso Jaramillo
Engineering CRISPR guide RNA riboswitches for in vivo applications Journal Article
In: Current Opinion in Biotechnology, vol. 55, pp. 103–113, 2019, (9 citations (Semantic Scholar/DOI) [2021-05-15]).
Links | BibTeX | Altmetric | Tags:
@article{galizi_engineering_2019,
title = {Engineering CRISPR guide RNA riboswitches for in vivo applications},
author = {Roberto Galizi and Alfonso Jaramillo},
doi = {10.1016/j.copbio.2018.08.007},
year = {2019},
date = {2019-01-01},
urldate = {2019-01-01},
journal = {Current Opinion in Biotechnology},
volume = {55},
pages = {103--113},
note = {9 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2018
Teresa Cordero; Arantxa Rosado; Eszter Majer; Alfonso Jaramillo; Guillermo Rodrigo; José-Antonio Daròs
Boolean Computation in Plants Using Post-translational Genetic Control and a Visual Output Signal Journal Article
In: ACS Synthetic Biology, vol. 7, no. 10, pp. 2322–2330, 2018, (3 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{cordero_boolean_2018,
title = {Boolean Computation in Plants Using Post-translational Genetic Control and a Visual Output Signal},
author = {Teresa Cordero and Arantxa Rosado and Eszter Majer and Alfonso Jaramillo and Guillermo Rodrigo and José-Antonio Daròs},
doi = {10.1021/acssynbio.8b00214},
year = {2018},
date = {2018-01-01},
urldate = {2018-01-01},
journal = {ACS Synthetic Biology},
volume = {7},
number = {10},
pages = {2322--2330},
abstract = {Due to autotrophic growing capacity and extremely rich secondary metabolism, plants should be preferred targets of synthetic biology. However, developments in plants usually run below those in other taxonomic groups. In this work we engineered genetic circuits capable of logic YES, OR and AND Boolean computation in plant tissues with a visual output signal. The circuits, which are deployed by means of Agrobacterium tumefaciens, perform with the conditional activity of the MYB transcription factor Rosea1 from Antirrhinum majus inducing the accumulation of anthocyanins, plant endogenous pigments that are directly visible to the naked eye or accurately quantifiable by spectrophotometric analysis. The translational fusion of Rosea1 to several viral proteins, such as potyvirus NIb or fragments thereof, rendered the transcription factor inactive. However, anthocyanin accumulation could be restored by inserting protease cleavage sites between both moieties of the fusion and by coexpressing specific proteases, such as potyvirus nuclear inclusion a protease.},
note = {3 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Andreas K. Broedel; Mark Isalan; Alfonso Jaramillo
Engineering of biomolecules by bacteriophage directed evolution Journal Article
In: Current Opinion in Biotechnology, vol. 51, pp. 32–38, 2018, (10 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{broedel_engineering_2018,
title = {Engineering of biomolecules by bacteriophage directed evolution},
author = {Andreas K. Broedel and Mark Isalan and Alfonso Jaramillo},
doi = {10.1016/j.copbio.2017.11.004},
year = {2018},
date = {2018-01-01},
urldate = {2018-01-01},
journal = {Current Opinion in Biotechnology},
volume = {51},
pages = {32--38},
abstract = {Conventional in vivo directed evolution methods have primarily linked the biomolecule's activity to bacterial cell growth. Recent developments instead rely on the conditional growth of bacteriophages (phages), viruses that infect and replicate within bacteria. Here we review recent phage-based selection systems for in vivo directed evolution. These approaches have been applied to evolve a wide range of proteins including transcription factors, polymerases, proteases, DNA-binding proteins, and protein-protein interactions. Advances in this field expand the possible applications of protein and RNA engineering. This will ultimately result in new biomolecules with tailor-made properties, as well as giving us a better understanding of basic evolutionary processes.},
note = {10 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Eunice A. Ferreira; Catarina C. Pacheco; Filipe Pinto; José Pereira; Pedro Lamosa; Paulo Oliveira; Boris Kirov; Alfonso Jaramillo; Paula Tamagnini
Expanding the toolbox for Synechocystis sp. PCC 6803: validation of replicative vectors and characterization of a novel set of promoters Journal Article
In: Synthetic Biology, vol. 3, no. 1, pp. ysy014, 2018, (20 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{ferreira_expanding_2018,
title = {Expanding the toolbox for Synechocystis sp. PCC 6803: validation of replicative vectors and characterization of a novel set of promoters},
author = {Eunice A. Ferreira and Catarina C. Pacheco and Filipe Pinto and José Pereira and Pedro Lamosa and Paulo Oliveira and Boris Kirov and Alfonso Jaramillo and Paula Tamagnini},
doi = {10.1093/synbio/ysy014},
year = {2018},
date = {2018-01-01},
urldate = {2018-01-01},
journal = {Synthetic Biology},
volume = {3},
number = {1},
pages = {ysy014},
abstract = {Cyanobacteria are promising 'low-cost' cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO2 as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the 'green' Escherichia coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference P rnpB , three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the ggpS gene [encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol (GG)] and the synthetic device was introduced into Synechocystis using one of the SEVA plasmids. The presence of this device restored the production of the GG in a ggpS deficient mutant validating the functionality of the tools/device developed in this study.},
note = {20 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Catarina Coutinho Pacheco; Zsófia Büttel; Filipe Pinto; Guillermo Rodrigo; Javier Carrera; Alfonso Jaramillo; Paula Tamagnini
Modulation of intracellular O2 concentration in Escherichia coli strains using Oxygen Consuming Devices (OCDs) Journal Article
In: ACS Synthetic Biology, vol. 7, no. 7, pp. 1742–1752, 2018, (1 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{pacheco_modulation_2018,
title = {Modulation of intracellular O2 concentration in Escherichia coli strains using Oxygen Consuming Devices (OCDs)},
author = {Catarina Coutinho Pacheco and Zsófia Büttel and Filipe Pinto and Guillermo Rodrigo and Javier Carrera and Alfonso Jaramillo and Paula Tamagnini},
doi = {10.1021/acssynbio.7b00428},
year = {2018},
date = {2018-01-01},
urldate = {2018-01-01},
journal = {ACS Synthetic Biology},
volume = {7},
number = {7},
pages = {1742--1752},
abstract = {The use of cell factories for the production of bulk and value-added compounds is nowadays an advantageous alternative to the traditional methods. Nevertheless, the efficiency and productivity of several of these processes can improve with the implementation of microaerobic or anaerobic conditions. In the industrial set, laccases are appealing catalysts able to oxidize a wide range of substrates and reducing O2 to H2O. In this work, several laccase-based devices were designed and constructed foreseeing the modulation of intracellular oxygen concentration in bacterial chassis. These Oxygen Consuming Devices (OCDs) included Escherichia coli's native laccase (CueO) and three variants of this protein obtained by directed evolution. The OCDs were initially characterized in vitro in E. coli DH5α and subsequently in other E. coli strains and in vivo. Upon induction of the OCDs no major effect on growth was observed in four of the strains tested, and the analysis of the cell extracts protein profiles revealed increased levels of laccase. Moreover, oxygen consumption associated to the OCDs occurred in all conditions tested but the performance of the device was shown to be strain-dependent, highlighting the importance of the genetic background even in closely related strains/chassis. One of the laccase variants showed a 13- and 5-fold increase in oxidase activity and O2 consumption rates, respectively. Furthermore, it also possible to demonstrate O2 consumption in vivo using L-DOPA as substrate, which represents the proof of concept that these OCDs can be used for the generation of intracellular microaerobic/anaerobic environments. In addition, the modularity and orthogonality principles used for the development of these devices allow an easy reassembly and fine-tuning foreseeing their introduction into other chassis/systems.},
note = {1 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Anis Senoussi; Jonathan Lee Tin Wah; Yoshihiro Shimizu; Jerome Robert; Alfonso Jaramillo; Sven Findeiß; Ilka M. Axmann; André Estévez-Torres
Quantitative Characterization of Translational Riboregulators Using an in Vitro Transcription-Translation System Journal Article
In: ACS Synthetic Biology, vol. 7, no. 5, pp. 1269–1278, 2018, (8 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{senoussi_quantitative_2018,
title = {Quantitative Characterization of Translational Riboregulators Using an in Vitro Transcription-Translation System},
author = {Anis Senoussi and Jonathan Lee Tin Wah and Yoshihiro Shimizu and Jerome Robert and Alfonso Jaramillo and Sven Findeiß and Ilka M. Axmann and André Estévez-Torres},
doi = {10.1021/acssynbio.7b00387},
year = {2018},
date = {2018-01-01},
urldate = {2018-01-01},
journal = {ACS Synthetic Biology},
volume = {7},
number = {5},
pages = {1269--1278},
abstract = {Riboregulators are short RNA sequences that, upon binding to a ligand, change their secondary structure and influence the expression rate of a downstream gene. They constitute an attractive alternative to transcription factors for building synthetic gene regulatory networks because they can be engineered de novo. However, riboregulators are generally designed in silico and tested in vivo, which provides little quantitative information about their performances, thus hindering the improvement of design algorithms. Here we show that a cell-free transcription-translation (TX-TL) system provides valuable information about the performances of in silico designed riboregulators. We first propose a simple model that provides a quantitative definition of the dynamic range of a riboregulator. We further characterize two types of translational riboregulators composed of a cis-repressed (cr) and a trans-activating (ta) strand. At the DNA level we demonstrate that high concentrations of taDNA poisoned the activator until total shut off, in agreement with our model, and that relative dynamic ranges of riboregulators determined in vitro are in agreement with published in vivo data. At the RNA level, we show that this approach provides a fast and simple way to measure dissociation constants of functional riboregulators, in contrast to standard mobility-shift assays. Our method opens the route for using cell-free TX-TL systems for the quantitative characterization of functional riboregulators in order to improve their design in silico.},
note = {8 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Mark Howarth; Alfonso Jaramillo
ScienceDirect Editorial overview: Nanobiotechnology: Baby steps and giant strides towards molecular mastery Journal Article
In: Current Opinion in Biotechnology, vol. 51, pp. iv–vi, 2018, (0 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{howarth_sciencedirect_2018,
title = {ScienceDirect Editorial overview: Nanobiotechnology: Baby steps and giant strides towards molecular mastery},
author = {Mark Howarth and Alfonso Jaramillo},
doi = {10.1016/j.copbio.2018.05.001},
year = {2018},
date = {2018-01-01},
journal = {Current Opinion in Biotechnology},
volume = {51},
pages = {iv--vi},
abstract = {Current Opinion in Biotechnology, 51 (2018) iv-vi. doi:10.1016/j.copbio.2018.05.001},
note = {0 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2017
Alfonso Jaramillo
Engineered stable ecosystems Journal Article
In: Nature microbiology, vol. 2, no. 8, pp. 17119, 2017, (0 citations (Semantic Scholar/DOI) [2021-05-15]).
Links | BibTeX | Altmetric | Tags:
@article{jaramillo_engineered_2017,
title = {Engineered stable ecosystems},
author = {Alfonso Jaramillo},
doi = {10.1038/nmicrobiol.2017.119},
year = {2017},
date = {2017-01-01},
urldate = {2017-01-01},
journal = {Nature microbiology},
volume = {2},
number = {8},
pages = {17119},
note = {0 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Guillermo Rodrigo; Satya Prakash; Shensi Shen; Eszter Majer; José-Antonio Daròs; Alfonso Jaramillo
Model-based design of RNA hybridization networks implemented in living cells Journal Article
In: Nucleic Acids Research, vol. 45, no. 16, pp. 9797–9808, 2017, (8 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{rodrigo_model-based_2017,
title = {Model-based design of RNA hybridization networks implemented in living cells},
author = {Guillermo Rodrigo and Satya Prakash and Shensi Shen and Eszter Majer and José-Antonio Daròs and Alfonso Jaramillo},
doi = {10.1093/nar/gkx698},
year = {2017},
date = {2017-01-01},
urldate = {2017-01-01},
journal = {Nucleic Acids Research},
volume = {45},
number = {16},
pages = {9797--9808},
abstract = {Synthetic gene circuits allow the behavior of living cells to be reprogrammed, and non-coding small RNAs (sRNAs) are increasingly being used as programmable regulators of gene expression. However, sRNAs (natural or synthetic) are generally used to regulate single target genes, while complex dynamic behaviors would require networks of sRNAs regulating each other. Here, we report a strategy for implementing such networks that exploits hybridization reactions carried out exclusively by multifaceted sRNAs that are both targets of and triggers for other sRNAs. These networks are ultimately coupled to the control of gene expression. We relied on a thermodynamic model of the different stable conformational states underlying this system at the nucleotide level. To test our model, we designed five different RNA hybridization networks with a linear architecture, and we implemented them in Escherichia coli. We validated the network architecture at the molecular level by native polyacrylamide gel electrophoresis, as well as the network function at the bacterial population and single-cell levels with a fluorescent reporter. Our results suggest that it is possible to engineer complex cellular programs based on RNA from first principles. Because these networks are mainly based on physical interactions, our designs could be expanded to other organisms as portable regulatory resources or to implement biological computations.},
note = {8 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Andreas K. Broedel; Alfonso Jaramillo; Mark Isalan
Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication Journal Article
In: Nature Protocols, vol. 12, no. 9, pp. 1830–1843, 2017, (12 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{broedel_intracellular_2017,
title = {Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication},
author = {Andreas K. Broedel and Alfonso Jaramillo and Mark Isalan},
url = {http://doi.org/10.1038/nprot.2017.084},
doi = {10.1038/nprot.2017.084},
year = {2017},
date = {2017-01-01},
urldate = {2017-01-01},
journal = {Nature Protocols},
volume = {12},
number = {9},
pages = {1830--1843},
abstract = {Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. These include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. A typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (PM) library expressing variants of the gene of interest (GOI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. The system has been developed for the selection of new orthogonal transcription factors (TFs) but is capable of evolving any gene-or gene circuit function-that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage λ cI TF against two synthetic bidirectional promoters. The evolved TF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. This protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ∼7 weeks.},
note = {12 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2016
Andreas K. Brdel; Alfonso Jaramillo; Mark Isalan
Engineering orthogonal dual transcription factors for multi-input synthetic promoters. Journal Article
In: Nature Communications, vol. 7, pp. 13858, 2016.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{brdel_engineering_2016,
title = {Engineering orthogonal dual transcription factors for multi-input synthetic promoters.},
author = {Andreas K. Brdel and Alfonso Jaramillo and Mark Isalan},
doi = {10.1038/ncomms13858},
year = {2016},
date = {2016-01-01},
urldate = {2016-01-01},
journal = {Nature Communications},
volume = {7},
pages = {13858},
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 $textbackslashlambda$ 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Guillermo Rodrigo; Satya Prakash; Teresa Cordero; Manish Kushwaha; Alfonso Jaramillo
Functionalization of an Antisense Small RNA Journal Article
In: Journal of Molecular Biology, vol. 428, no. 5 Pt B, pp. 889–892, 2016, (4 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{rodrigo_functionalization_2016,
title = {Functionalization of an Antisense Small RNA},
author = {Guillermo Rodrigo and Satya Prakash and Teresa Cordero and Manish Kushwaha and Alfonso Jaramillo},
doi = {10.1016/j.jmb.2015.12.022},
year = {2016},
date = {2016-01-01},
urldate = {2016-01-01},
journal = {Journal of Molecular Biology},
volume = {428},
number = {5 Pt B},
pages = {889--892},
abstract = {In order to explore the possibility of adding new functions to preexisting genes, we considered a framework of riboregulation. We created a new riboregulator consisting of the reverse complement of a known riboregulator. Using computational design, we engineered a cis-repressing 5' untranslated region that can be activated by this new riboregulator. As a result, both RNAs can orthogonally trans-activate translation of their cognate, independent targets. The two riboregulators can also repress each other by antisense interaction, although not symmetrically. Our work highlights that antisense small RNAs can work as regulatory agents beyond the antisense paradigm and that, hence, they could be interfaced with other circuits used in synthetic biology.},
note = {4 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Antonia P. Sagona; Aurelija M. Grigonyte; Paul R. MacDonald; Alfonso Jaramillo
Genetically modified bacteriophages Journal Article
In: Integrative biology : quantitative biosciences from nano to macro, vol. 8, no. 4, pp. 465–474, 2016, (17 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{sagona_genetically_2016,
title = {Genetically modified bacteriophages},
author = {Antonia P. Sagona and Aurelija M. Grigonyte and Paul R. MacDonald and Alfonso Jaramillo},
doi = {10.1039/c5ib00267b},
year = {2016},
date = {2016-01-01},
urldate = {2016-01-01},
journal = {Integrative biology : quantitative biosciences from nano to macro},
volume = {8},
number = {4},
pages = {465--474},
abstract = {Phages or bacteriophages, viruses that infect and replicate inside bacteria, are the most abundant microorganisms on earth. The realization that antibiotic resistance poses a substantial risk to the world's health and global economy is revitalizing phage therapy as a potential solution. The increasing ease by which phage genomes can be modified, owing to the influx of new technologies, has led to an expansion of their natural capabilities, and a reduced dependence on phage isolation from environmental sources. This review will discuss the way synthetic biology has accelerated the construction of genetically modified phages and will describe the wide range of their applications. It will further provide insight into the societal and economic benefits that derive from the use of recombinant phages in various sectors, from health to biodetection, biocontrol and the food industry.},
note = {17 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Manish Kushwaha; William Rostain; Satya Prakash; John N. Duncan; Alfonso Jaramillo
Using RNA as Molecular Code for Programming Cellular Function Journal Article
In: ACS Synthetic Biology, vol. 5, no. 8, pp. 795–809, 2016, (32 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{kushwaha_using_2016,
title = {Using RNA as Molecular Code for Programming Cellular Function},
author = {Manish Kushwaha and William Rostain and Satya Prakash and John N. Duncan and Alfonso Jaramillo},
doi = {10.1021/acssynbio.5b00297},
year = {2016},
date = {2016-01-01},
urldate = {2016-01-01},
journal = {ACS Synthetic Biology},
volume = {5},
number = {8},
pages = {795--809},
abstract = {RNA is involved in a wide-range of important molecular processes in the cell, serving diverse functions: regulatory, enzymatic, and structural. Together with its ease and predictability of design, these properties can lead RNA to become a useful handle for biological engineers with which to control the cellular machinery. By modifying the many RNA links in cellular processes, it is possible to reprogram cells toward specific design goals. We propose that RNA can be viewed as a molecular programming language that, together with protein-based execution platforms, can be used to rewrite wide ranging aspects of cellular function. In this review, we catalogue developments in the use of RNA parts, methods, and associated computational models that have contributed to the programmability of biology. We discuss how RNA part repertoires have been combined to build complex genetic circuits, and review recent applications of RNA-based parts and circuitry. We explore the future potential of RNA engineering and posit that RNA programmability is an important resource for firmly establishing an era of rationally designed synthetic biology.},
note = {32 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Martina Pasini; Alfred Fernández-Castané; Alfonso Jaramillo; Carles Mas; Gloria Caminal; Pau Ferrer
Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli Journal Article
In: New biotechnology, vol. 33, no. 1, pp. 78–90, 2016, (20 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{pasini_using_2016,
title = {Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli},
author = {Martina Pasini and Alfred Fernández-Castané and Alfonso Jaramillo and Carles Mas and Gloria Caminal and Pau Ferrer},
doi = {10.1016/j.nbt.2015.08.003},
year = {2016},
date = {2016-01-01},
urldate = {2016-01-01},
journal = {New biotechnology},
volume = {33},
number = {1},
pages = {78--90},
abstract = {The over-expression of proteins in recombinant host cells often requires a significant amount of resources causing an increase in the metabolic load for the host. This results in a variety of physiological responses leading to altered growth parameters, including growth inhibition or activation of secondary metabolism pathways. Moreover, the expression of other plasmid-encoded genes such as antibiotic resistance genes or repressor proteins may also alter growth kinetics. In this work, we have developed a second-generation system suitable for Escherichia coli expression with an antibiotic-free plasmid maintenance mechanism based on a glycine auxotrophic marker (glyA). Metabolic burden related to plasmid maintenance and heterologous protein expression was minimized by tuning the expression levels of the repressor protein (LacI) and glyA using a library of promoters and applying synthetic biology tools that allow the rapid construction of vectors. The engineered antibiotic-free expression system was applied to the L-fuculose phosphate aldolase (FucA) over-production, showing an increase in production up to 3.8-fold in terms of FucA yield (mg g(-1)DCW) and 4.5-fold in terms of FucA activity (AU g(-1)DCW) compared to previous expression. Moreover, acetic acid production was reduced to 50%, expressed as gAc gDCW(-1). Our results showed that the aforementioned approaches are of paramount importance in order to increment the protein production in terms of mass and activity.},
note = {20 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2015
Martyn Amos; Ilka Maria Axmann; Nils Bluthgen; Fernando Cruz; Alfonso Jaramillo; Alfonso Rodriguez-Paton; Friedrich Simmel
Bacterial computing with engineered populations Journal Article
In: vol. 373, no. 2046, pp. 20140218, 2015, (11 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{amos_bacterial_2015,
title = {Bacterial computing with engineered populations},
author = {Martyn Amos and Ilka Maria Axmann and Nils Bluthgen and Fernando Cruz and Alfonso Jaramillo and Alfonso Rodriguez-Paton and Friedrich Simmel},
doi = {10.1098/rsta.2014.0218},
year = {2015},
date = {2015-01-01},
volume = {373},
number = {2046},
pages = {20140218},
abstract = {We describe strategies for the construction of bacterial computing platforms by describing a number of results from the recently completed bacterial computing with engineered populations project. In general, the implementation of such systems requires a framework containing various components such as intracellular circuits, single cell input/output and cell-cell interfacing, as well as extensive analysis. In this overview paper, we describe our approach to each of these, and suggest possible areas for future research.},
note = {11 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Jack Hassall; Paul MacDonald; Teresa Cordero; William Rostain; Alfonso Jaramillo
Design and Characterization of Topological Small RNAs Journal Article
In: Methods in molecular biology (Clifton, N.J.), vol. 1316, no. Chapter 13, pp. 149–167, 2015, ISSN: 978-1-4939-2729-6, (1 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{hassall_design_2015,
title = {Design and Characterization of Topological Small RNAs},
author = {Jack Hassall and Paul MacDonald and Teresa Cordero and William Rostain and Alfonso Jaramillo},
doi = {10.1007/978-1-4939-2730-2_13},
issn = {978-1-4939-2729-6},
year = {2015},
date = {2015-01-01},
urldate = {2015-01-01},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1316},
number = {Chapter 13},
pages = {149--167},
abstract = {RNA can self-assemble into complex structures through base pairing, as well as encode information and bind with proteins to induce enzymatic activity. Furthermore, RNA can possess intrinsic enzymatic-like (ribozymatic) activity, a property that, if necessary, can be activated only upon the binding of a small molecule or another RNA (as is the case in aptazymes). As such, RNA could be of use in nanotechnology as a programmable polymer capable of self-assembling into complex topological structures. In this chapter we describe a method for designing advanced topological structures using self-circulating RNA, exemplified by three tiers of topologically manipulated self-assembling synthetic RNA systems. The first tier of topological manipulation, the RNA knot is a physically locked structure, formed by circularizing one monomer of knotted single-stranded RNA left with loose ends (an "open" knot). The second tier, a two interlocking ring system, is made by interlocking two circular RNA components: a circular RNA target, and an RNA lasso designed to intercalate the target before circularizing. The third tier naturally extends this system into a string of topologically locked circular RNA molecules (an RNA chain). We detail the methodology used for designing such topologically complex RNAs, including computational predictions of secondary structure, and where appropriate, RNA-RNA interactions, illustrated by examples. We then describe the experimental methods used for characterizing such structures, and provide sequences of building blocks that can be used for topological manipulation of RNA.},
note = {1 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Alfonso Jaramillo
Droplet-based method and device for the continuous proliferation of living cells in suspension Miscellaneous
2015.
BibTeX | Tags:
@misc{Alfonso_Jaramillo_102243168,
title = {Droplet-based method and device for the continuous proliferation of living cells in suspension},
author = {Alfonso Jaramillo},
year = {2015},
date = {2015-01-01},
keywords = {},
pubstate = {published},
tppubtype = {misc}
}
Shensi Shen; Guillermo Rodrigo; Satya Prakash; Eszter Majer; Thomas E. Landrain; Boris Kirov; Jos-Antonio Dars; Alfonso Jaramillo
Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression. Journal Article
In: Nucleic Acids Research, vol. 43, no. 10, pp. 5158–5170, 2015.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{shen_dynamic_2015,
title = {Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression.},
author = {Shensi Shen and Guillermo Rodrigo and Satya Prakash and Eszter Majer and Thomas E. Landrain and Boris Kirov and Jos-Antonio Dars and Alfonso Jaramillo},
doi = {10.1093/nar/gkv287},
year = {2015},
date = {2015-01-01},
urldate = {2015-01-01},
journal = {Nucleic Acids Research},
volume = {43},
number = {10},
pages = {5158--5170},
abstract = {Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA-RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Guillermo Rodrigo; Eszter Majer; Satya Prakash; Jos-Antonio Dars; Alfonso Jaramillo; Juan F. Poyatos
Exploring the dynamics and mutational landscape of riboregulation with a minimal synthetic circuit in living cells Journal Article
In: Biophysical Journal, vol. 109, pp. 1070–1076, 2015.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{rodrigo_exploring_2015,
title = {Exploring the dynamics and mutational landscape of riboregulation with a minimal synthetic circuit in living cells},
author = {Guillermo Rodrigo and Eszter Majer and Satya Prakash and Jos-Antonio Dars and Alfonso Jaramillo and Juan F. Poyatos},
doi = {10.1016/j.bpj.2015.07.021},
year = {2015},
date = {2015-01-01},
journal = {Biophysical Journal},
volume = {109},
pages = {1070--1076},
abstract = {Regulation of gene expression triggered by conformational changes in RNA molecules is widely observed in cellular systems. Here, we examine this mode of control by means of a model-based de- sign and construction of a fully synthetic riboregulatory device. We present a theoretical framework that rests on a simple energy model to predict the dynamic response of such a system. Following an equilibrium description, our framework integrates thermodynamic properties textbackslashtextendash anticipated with an RNA physicochemical model textbackslashtextendash with a detailed description of the intermolecular interaction. The theoretical calculations are confirmed with an experimental characterization of the action of the riboregulatory device within living cells. This illustrates, more broadly, the predictability of ge- netic robustness on synthetic systems, and the faculty to engineer gene expression programs from a minimal set of first principles.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
William Rostain; Thomas E. Landrain; Guillermo Rodrigo; Alfonso Jaramillo
Regulatory RNA design through evolutionary computation and strand displacement Journal Article
In: Methods in molecular biology (Clifton, N.J.), vol. 1244, pp. 63–78, 2015.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{rostain_regulatory_2015,
title = {Regulatory RNA design through evolutionary computation and strand displacement},
author = {William Rostain and Thomas E. Landrain and Guillermo Rodrigo and Alfonso Jaramillo},
doi = {10.1007/978-1-4939-1878-2_4},
year = {2015},
date = {2015-01-01},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1244},
pages = {63--78},
abstract = {The discovery and study of a vast number of regulatory RNAs in all kingdoms of life over the past decades has allowed the design of new synthetic RNAs that can regulate gene expression in vivo. Riboregulators, in particular, have been used to activate or repress gene expression. However, to accelerate and scale up the design process, synthetic biologists require computer-assisted design tools, without which riboregulator engineering will remain a case-by-case design process requiring expert attention. Recently, the design of RNA circuits by evolutionary computation and adapting strand displacement techniques from nanotechnology has proven to be suited to the automated generation of DNA sequences implementing regulatory RNA systems in bacteria. Herein, we present our method to carry out such evolutionary design and how to use it to create various types of riboregulators, allowing the systematic de novo design of genetic control systems in synthetic biology.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2014
P. Parutto; L. Le Goff; H. Léger; G. Mercy; E. Kouwe; A. Chhun; B. Baudu; G. Guillocheau; F. Amiot; L. Ujéda; W. Rostain; C. Pauthenier; T. Cerisy; N. Pollet; A. Tolonen; Alfonso Jaramillo
Characterization and modeling of an iron-sensitive system in Escherichia coli Inproceedings
In: pp. 107–118, 2014.
@inproceedings{parutto_characterization_2014-1,
title = {Characterization and modeling of an iron-sensitive system in Escherichia coli},
author = {P. Parutto and L. Le Goff and H. Léger and G. Mercy and E. Kouwe and A. Chhun and B. Baudu and G. Guillocheau and F. Amiot and L. Ujéda and W. Rostain and C. Pauthenier and T. Cerisy and N. Pollet and A. Tolonen and Alfonso Jaramillo},
url = {http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.686.1367&rep=rep1&type=pdf#page=107},
year = {2014},
date = {2014-07-01},
urldate = {2014-07-01},
pages = {107--118},
keywords = {},
pubstate = {published},
tppubtype = {inproceedings}
}
Guillermo Rodrigo; Satya Prakash; Shensi Shen; Eszter Majer; Jos-Antonio Dars; Alfonso Jaramillo
Cooperative riboregulation in living cells through allosteric programming of toehold activation Journal Article
In: BioRxiv, pp. 009688, 2014.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{rodrigo_cooperative_2014,
title = {Cooperative riboregulation in living cells through allosteric programming of toehold activation},
author = {Guillermo Rodrigo and Satya Prakash and Shensi Shen and Eszter Majer and Jos-Antonio Dars and Alfonso Jaramillo},
doi = {10.1101/009688},
year = {2014},
date = {2014-01-01},
journal = {BioRxiv},
pages = {009688},
abstract = {Abstract Living cells rely on small non-coding RNAs (sRNAs) to regulate gene expression at the post-transcriptional level. Contrary to most protein-based activators of transcription, all known riboregulators do not exploit cooperative binding mechanisms to activate gene ...},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
William Rostain; Shensi Shen; Teresa Cordero; Guillermo Rodrigo; Alfonso Jaramillo
Engineering a circular riboregulator in Escherichia coli Journal Article
In: BioRxiv, pp. 008987, 2014.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{rostain_engineering_2014,
title = {Engineering a circular riboregulator in Escherichia coli},
author = {William Rostain and Shensi Shen and Teresa Cordero and Guillermo Rodrigo and Alfonso Jaramillo},
doi = {10.1101/008987},
year = {2014},
date = {2014-01-01},
journal = {BioRxiv},
pages = {008987},
abstract = {Abstract Circular RNAs have recently been shown to be important gene expression regulators in mammalian cells. However, their role in prokaryotes remains elusive. Here, we engineered a synthetic riboregulator that self-splice to produce a circular molecule, ...},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Guillermo Rodrigo; Alfonso Jaramillo
RiboMaker: computational design of conformation-based riboregulation Journal Article
In: Bioinformatics, vol. 30, no. 17, pp. 2508–2510, 2014.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{rodrigo_ribomaker_2014,
title = {RiboMaker: computational design of conformation-based riboregulation},
author = {Guillermo Rodrigo and Alfonso Jaramillo},
doi = {10.1093/bioinformatics/btu335},
year = {2014},
date = {2014-01-01},
journal = {Bioinformatics},
volume = {30},
number = {17},
pages = {2508--2510},
abstract = {MOTIVATION:The ability to engineer control systems of gene expression is instrumental for synthetic biology. Thus, bioinformatic methods that assist such engineering are appealing because they can guide the sequence design and prevent costly experimental screening. In particular, RNA is an ideal substrate to de novo design regulators of protein expression by following sequence-to-function models. RESULTS:We have implemented a novel algorithm, RiboMaker, aimed at the computational, automated design of bacterial riboregulation. RiboMaker reads the sequence and structure specifications, which codify for a gene regulatory behaviour, and optimizes the sequences of a small regulatory RNA and a 5'-untranslated region for an efficient intermolecular interaction. To this end, it implements an evolutionary design strategy, where random mutations are selected according to a physicochemical model based on free energies. The resulting sequences can then be tested experimentally, providing a new tool for synthetic biology, and also for investigating the riboregulation principles in natural systems. AVAILABILITY AND IMPLEMENTATION:Web server is available at http://ribomaker.jaramillolab.org/. Source code, instructions and examples are freely available for download at http://sourceforge.net/projects/ribomaker/.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2013
Guillermo Rodrigo; Thomas E. Landrain; Shensi Shen; Alfonso Jaramillo
A new frontier in synthetic biology: automated design of small RNA devices in bacteria Journal Article
In: Trends in genetics : TIG, vol. 29, no. 9, pp. 529–536, 2013.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{rodrigo_new_2013,
title = {A new frontier in synthetic biology: automated design of small RNA devices in bacteria},
author = {Guillermo Rodrigo and Thomas E. Landrain and Shensi Shen and Alfonso Jaramillo},
doi = {10.1016/j.tig.2013.06.005},
year = {2013},
date = {2013-01-01},
journal = {Trends in genetics : TIG},
volume = {29},
number = {9},
pages = {529--536},
abstract = {RNA devices provide synthetic biologists with tools for manipulating post-transcriptional regulation and conditional detection of cellular biomolecules. The use of computational methods to design RNA devices has improved to the stage where it is now possible to automate the entire design process. These methods utilize structure prediction tools that optimize nucleotide sequences, together with fragments of known independent functionalities. Recently, this approach has been used to create an automated method for the de novo design of riboregulators. Here, we describe how it is possible to obtain riboregulatory circuits in prokaryotes by capturing the relevant interactions of RNAs inside the cytoplasm using a physicochemical model. We focus on the regulation of protein expression mediated by intra- or intermolecular interactions of small RNAs (sRNAs), and discuss the design of riboregulators for other functions. The automated design of RNA devices opens new possibilities for engineering fully synthetic regulatory systems that program new functions or reprogram dysfunctions in living cells.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Guillermo Rodrigo; Alfonso Jaramillo
AutoBioCAD: full biodesign automation of genetic circuits Journal Article
In: ACS Synthetic Biology, vol. 2, no. 5, pp. 230–236, 2013.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{rodrigo_autobiocad_2013,
title = {AutoBioCAD: full biodesign automation of genetic circuits},
author = {Guillermo Rodrigo and Alfonso Jaramillo},
doi = {10.1021/sb300084h},
year = {2013},
date = {2013-01-01},
journal = {ACS Synthetic Biology},
volume = {2},
number = {5},
pages = {230--236},
abstract = {Synthetic regulatory networks with prescribed functions are engineered by assembling a reduced set of functional elements. We could also assemble them computationally if the mathematical models of those functional elements were predictive enough in different genetic contexts. Only after achieving this will we have libraries of models of biological parts able to provide predictive dynamical behaviors for most circuits constructed with them. We thus need tools that can automatically explore different genetic contexts, in addition to being able to use such libraries to design novel circuits with targeted dynamics. We have implemented a new tool, AutoBioCAD, aimed at the automated design of gene regulatory circuits. AutoBioCAD loads a library of models of genetic elements and implements evolutionary design strategies to produce (i) nucleotide sequences encoding circuits with targeted dynamics that can then be tested experimentally and (ii) circuit models for testing regulation principles in natural systems, providing a new tool for synthetic biology. AutoBioCAD can be used to model and design genetic circuits with dynamic behavior, thanks to the incorporation of stochastic effects, robustness, qualitative dynamics, multiobjective optimization, or degenerate nucleotide sequences, all facilitating the link with biological part/circuit engineering.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Javier Carrera; Alfonso Jaramillo
Automated design of bacterial genome sequences Journal Article
In: BMC systems biology, vol. 7, pp. 108, 2013, (1 citations (Semantic Scholar/DOI) [2021-05-15]).
Abstract | Links | BibTeX | Altmetric | Tags:
@article{carrera_automated_2013,
title = {Automated design of bacterial genome sequences},
author = {Javier Carrera and Alfonso Jaramillo},
doi = {10.1186/1752-0509-7-108},
year = {2013},
date = {2013-01-01},
journal = {BMC systems biology},
volume = {7},
pages = {108},
abstract = {BACKGROUND:Organisms have evolved ways of regulating transcription to better adapt to varying environments. Could the current functional genomics data and models support the possibility of engineering a genome with completely rearranged gene organization while the cell maintains its behavior under environmental challenges? How would we proceed to design a full nucleotide sequence for such genomes? RESULTS:As a first step towards answering such questions, recent work showed that it is possible to design alternative transcriptomic models showing the same behavior under environmental variations than the wild-type model. A second step would require providing evidence that it is possible to provide a nucleotide sequence for a genome encoding such transcriptional model. We used computational design techniques to design a rewired global transcriptional regulation of Escherichia coli, yet showing a similar transcriptomic response than the wild-type. Afterwards, we "compiled" the transcriptional networks into nucleotide sequences to obtain the final genome sequence. Our computational evolution procedure ensures that we can maintain the genotype-phenotype mapping during the rewiring of the regulatory network. We found that it is theoretically possible to reorganize E. coli genome into 86% fewer regulated operons. Such refactored genomes are constituted by operons that contain sets of genes sharing around the 60% of their biological functions and, if evolved under highly variable environmental conditions, have regulatory networks, which turn out to respond more than 20% faster to multiple external perturbations. CONCLUSIONS:This work provides the first algorithm for producing a genome sequence encoding a rewired transcriptional regulation with wild-type behavior under alternative environments.},
note = {1 citations (Semantic Scholar/DOI) [2021-05-15]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Mara Surez Diez; Anas M. Pujol; Manolis Matzapetakis; Alfonso Jaramillo; Olga Iranzo
In: Biotechnology Journal, vol. 8, no. 7, pp. 855–864, 2013.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{surez_diez_computational_2013,
title = {Computational protein design with electrostatic focusing: experimental characterization of a conditionally folded helical domain with a reduced amino acid alphabet.},
author = {Mara Surez Diez and Anas M. Pujol and Manolis Matzapetakis and Alfonso Jaramillo and Olga Iranzo},
doi = {10.1002/biot.201200380},
year = {2013},
date = {2013-01-01},
journal = {Biotechnology Journal},
volume = {8},
number = {7},
pages = {855--864},
abstract = {Automated methodologies to design synthetic proteins from first principles use energy computations to estimate the ability of the sequences to adopt a targeted structure. This approach is still far from systematically producing native-like sequences, due, most likely, to inaccuracies when modeling the interactions between the protein and its aqueous environment. This is particularly challenging when engineering small protein domains (with less polar pair interactions than with the solvent). We have re-designed a three-helix bundle, domain B, using a fixed backbone and a four amino acid alphabet. We have enlarged the rotamer library with conformers that increase the weight of electrostatic interactions within the design process without altering the energy function used to compute the folding free energy. Our synthetic sequences show less than 15% similarity to any Swissprot sequence. We have characterized our sequences in different solvents using circular dichroism and nuclear magnetic resonance. The targeted structure achieved is dependent on the solvent used. This method can be readily extended to larger domains. Our method will be useful for the engineering of proteins that become active only in a given solvent and for designing proteins in the context of hydrophobic solvents, an important fraction of the situations in the cell.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A. Jaramillo; D. Zeliszewski
Design of the living automates | La conception du vivant s'automatise Journal Article
In: Biofutur, no. 339, pp. 36-39, 2013.
BibTeX | Tags:
@article{jaramillo2013,
title = {Design of the living automates | La conception du vivant s'automatise},
author = {A. Jaramillo and D. Zeliszewski},
year = {2013},
date = {2013-01-01},
journal = {Biofutur},
number = {339},
pages = {36-39},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Guillermo Rodrigo; Thomas E. Landrain; Eszter Majer; Jos-Antonio Dars; Alfonso Jaramillo
Full Design Automation of Multi-State RNA Devices to Program Gene Expression Using Energy-Based Optimization Journal Article
In: PLoS Computational Biology, vol. 9, no. 8, pp. e1003172, 2013.
Links | BibTeX | Altmetric | Tags:
@article{rodrigo_full_2013,
title = {Full Design Automation of Multi-State RNA Devices to Program Gene Expression Using Energy-Based Optimization},
author = {Guillermo Rodrigo and Thomas E. Landrain and Eszter Majer and Jos-Antonio Dars and Alfonso Jaramillo},
doi = {10.1371/journal.pcbi.1003172.s009},
year = {2013},
date = {2013-01-01},
journal = {PLoS Computational Biology},
volume = {9},
number = {8},
pages = {e1003172},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Alfonso Jaramillo; Alfonso Jaramillo; Dominique Zeliszewski
The idea of automated living Journal Article
In: Biofutur, 2013.
BibTeX | Tags:
@article{Alfonso_Jaramillo_102297198,
title = {The idea of automated living},
author = {Alfonso Jaramillo and Alfonso Jaramillo and Dominique Zeliszewski},
year = {2013},
date = {2013-01-01},
journal = {Biofutur},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Guillermo Rodrigo; Boris Kirov; Shensi Shen; Alfonso Jaramillo
Theoretical and experimental analysis of the forced LacI-AraC oscillator with a minimal gene regulatory model Journal Article
In: Chaos: An Interdisciplinary Journal of Nonlinear Science, vol. 23, no. 2, pp. 025109, 2013.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{rodrigo_theoretical_2013,
title = {Theoretical and experimental analysis of the forced LacI-AraC oscillator with a minimal gene regulatory model},
author = {Guillermo Rodrigo and Boris Kirov and Shensi Shen and Alfonso Jaramillo},
doi = {10.1063/1.4809786},
year = {2013},
date = {2013-01-01},
journal = {Chaos: An Interdisciplinary Journal of Nonlinear Science},
volume = {23},
number = {2},
pages = {025109},
abstract = {Oscillatory dynamics have been observed in multiple cellular functions and synthetic constructs; and here, we study the behavior of a synthetic oscillator under temporal perturbations. We use a minimal model, involving a single transcription factor with delayed self-repression and enzymatic degradation, together with a first-order perturbative approach, to derive an analytical expression for the power spectrum of the system, which characterizes its response to external forces and molecular noise. Experimentally, we force and monitor the dynamics of the LacI-AraC oscillator in single cells during long time intervals by constructing a microfluidics device. Pulse dynamics of IPTG with different periods serve to perturb this system. Due to the resonance of the system, we predict theoretically and confirm experimentally the dependence on the forcing frequency of the variability in gene expression with time and the synchronization of the population to the input signal. The reported results show that the engineering of gene circuits can provide test cases for dynamical models, which could be further exploited in synthetic biology.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2012
Catalin I. Fetita; Boris Kirov; Alfonso Jaramillo; Christophe Lefevre
An automated approach for single-cell tracking in epifluorescence microscopy applied to E. coli growth analysis on microfluidics biochips Inproceedings
In: Medical Imaging, 2012.
BibTeX | Tags:
@inproceedings{Fetita2012AnAA,
title = {An automated approach for single-cell tracking in epifluorescence microscopy applied to E. coli growth analysis on microfluidics biochips},
author = {Catalin I. Fetita and Boris Kirov and Alfonso Jaramillo and Christophe Lefevre},
year = {2012},
date = {2012-01-01},
booktitle = {Medical Imaging},
keywords = {},
pubstate = {published},
tppubtype = {inproceedings}
}
Javier Carrera; J. Carrera; S. F. Elena; Santiago F. Elena; Alfonso Jaramillo
Computational design of genomic transcriptional networks with adaptation to varying environments. Journal Article
In: Proceedings of the National Academy of Sciences, vol. 109, no. 38, pp. 15277–15282, 2012.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{carrera_computational_2012,
title = {Computational design of genomic transcriptional networks with adaptation to varying environments.},
author = {Javier Carrera and J. Carrera and S. F. Elena and Santiago F. Elena and Alfonso Jaramillo},
doi = {10.1073/pnas.1200030109},
year = {2012},
date = {2012-01-01},
journal = {Proceedings of the National Academy of Sciences},
volume = {109},
number = {38},
pages = {15277--15282},
abstract = {Transcriptional profiling has been widely used as a tool for unveiling the coregulations of genes in response to genetic and environmental perturbations. These coregulations have been used, in a few instances, to infer global transcriptional regulatory models. Here, using the large amount of transcriptomic information available for the bacterium Escherichia coli, we seek to understand the design principles determining the regulation of its transcriptome. Combining transcriptomic and signaling data, we develop an evolutionary computational procedure that allows obtaining alternative genomic transcriptional regulatory network (GTRN) that still maintains its adaptability to dynamic environments. We apply our methodology to an E. coli GTRN and show that it could be rewired to simpler transcriptional regulatory structures. These rewired GTRNs still maintain the global physiological response to fluctuating environments. Rewired GTRNs contain 73% fewer regulated operons. Genes with similar functions and coordinated patterns of expression across environments are clustered into longer regulated operons. These synthetic GTRNs are more sensitive and show a more robust response to challenging environments. This result illustrates that the natural configuration of E. coli GTRN does not necessarily result from selection for robustness to environmental perturbations, but that evolutionary contingencies may have been important as well. We also discuss the limitations of our methodology in the context of the demand theory. Our procedure will be useful as a novel way to analyze global transcription regulation networks and in synthetic biology for the de novo design of genomes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Guillermo Rodrigo; Thomas E. Landrain; Alfonso Jaramillo
De novo automated design of small RNA circuits for engineering synthetic riboregulation in living cells Journal Article
In: Proceedings of the National Academy of Sciences of the United States of America, vol. 109, no. 38, pp. 15271–15276, 2012.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{rodrigo_novo_2012,
title = {De novo automated design of small RNA circuits for engineering synthetic riboregulation in living cells},
author = {Guillermo Rodrigo and Thomas E. Landrain and Alfonso Jaramillo},
doi = {10.1073/pnas.1203831109},
year = {2012},
date = {2012-01-01},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {109},
number = {38},
pages = {15271--15276},
abstract = {A grand challenge in synthetic biology is to use our current knowledge of RNA science to perform the automatic engineering of completely synthetic sequences encoding functional RNAs in living cells. We report here a fully automated design methodology and experimental validation of synthetic RNA interaction circuits working in a cellular environment. The computational algorithm, based on a physicochemical model, produces novel RNA sequences by exploring the space of possible sequences compatible with predefined structures. We tested our methodology in Escherichia coli by designing several positive riboregulators with diverse structures and interaction models, suggesting that only the energy of formation and the activation energy (free energy barrier to overcome for initiating the hybridization reaction) are sufficient criteria to engineer RNA interaction and regulation in bacteria. The designed sequences exhibit nonsignificant similarity to any known noncoding RNA sequence. Our riboregulatory devices work independently and in combination with transcription regulation to create complex logic circuits. Our results demonstrate that a computational methodology based on first-principles can be used to engineer interacting RNAs with allosteric behavior in living cells.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
G. Rodrigo; A. Jaramillo; M. A. Blázquez
Erratum: Integral control of plant gravitropism through the interplay of hormone signaling and gene regulation (Biophysical Journal (2011) 101 (757-763)) Journal Article
In: Biophysical Journal, vol. 102, no. 10, 2012.
BibTeX | Tags:
@article{jaramillo2012,
title = {Erratum: Integral control of plant gravitropism through the interplay of hormone signaling and gene regulation (Biophysical Journal (2011) 101 (757-763))},
author = {G. Rodrigo and A. Jaramillo and M. A. Blázquez},
year = {2012},
date = {2012-01-01},
journal = {Biophysical Journal},
volume = {102},
number = {10},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Javier Carrera; Asun Fernndez Carmen; Rafael Fernndez-Muoz; Jose Luis Rambla; Clara Pons; Alfonso Jaramillo; Santiago F. Elena; Antonio Granell
Fine-tuning tomato agronomic properties by computational genome redesign. Journal Article
In: PLoS Computational Biology, vol. 8, no. 6, pp. e1002528, 2012.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{carrera_fine-tuning_2012,
title = {Fine-tuning tomato agronomic properties by computational genome redesign.},
author = {Javier Carrera and Asun Fernndez Carmen and Rafael Fernndez-Muoz and Jose Luis Rambla and Clara Pons and Alfonso Jaramillo and Santiago F. Elena and Antonio Granell},
doi = {10.1371/journal.pcbi.1002528.s014},
year = {2012},
date = {2012-01-01},
journal = {PLoS Computational Biology},
volume = {8},
number = {6},
pages = {e1002528},
abstract = {Considering cells as biofactories, we aimed to optimize its internal processes by using the same engineering principles that large industries are implementing nowadays: lean manufacturing. We have applied reverse engineering computational methods to transcriptomic, metabolomic and phenomic data obtained from a collection of tomato recombinant inbreed lines to formulate a kinetic and constraint-based model that efficiently describes the cellular metabolism from expression of a minimal core of genes. Based on predicted metabolic profiles, a close association with agronomic and organoleptic properties of the ripe fruit was revealed with high statistical confidence. Inspired in a synthetic biology approach, the model was used for exploring the landscape of all possible local transcriptional changes with the aim of engineering tomato fruits with fine-tuned biotechnological properties. The method was validated by the ability of the proposed genomes, engineered for modified desired agronomic traits, to recapitulate experimental correlations between associated metabolites.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Guillermo Rodrigo; Javier Carrera; Thomas E. Landrain; Alfonso Jaramillo
Perspectives on the automatic design of regulatory systems for synthetic biology Journal Article
In: FEBS Lett, vol. 586, no. 15, pp. 2037–2042, 2012.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{rodrigo_perspectives_2012,
title = {Perspectives on the automatic design of regulatory systems for synthetic biology},
author = {Guillermo Rodrigo and Javier Carrera and Thomas E. Landrain and Alfonso Jaramillo},
doi = {10.1016/j.febslet.2012.02.031},
year = {2012},
date = {2012-01-01},
journal = {FEBS Lett},
volume = {586},
number = {15},
pages = {2037--2042},
abstract = {Automatic design is based on computational modeling and optimization methods to provide prototype designs to targeted problems in an unsupervised manner. For biological circuits, we need to produce quantitative predictions of cell behavior for a given genotype as consequence of the different molecular interactions. Automatic design techniques aim at solving the inverse problem of finding the sequences of nucleotides that better fit a targeted behavior. In the post-genomic era, our molecular knowledge and modeling capabilities have allowed to start using such methodologies with success. Herein, we describe how the emergence of this new type of tools could enable novel synthetic biology applications. We highlight the essential elements to develop automatic design procedures for synthetic biology pointing out their advantages and bottlenecks. We discuss in detail the experimental difficulties to overcome in the in vivo implementation of designed networks. The use of automatic design to engineer biological networks is starting to emerge as a new technique to perform synthetic biology, which should not be neglected in the future.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2011
Guillermo Rodrigo; Javier Carrera; Alfonso Jaramillo
Computational design of synthetic regulatory networks from a genetic library to characterize the designability of dynamical behaviors Journal Article
In: Nucleic Acids Research, vol. 39, no. 20, pp. e138–e138, 2011.
Abstract | Links | BibTeX | Altmetric | Tags:
@article{rodrigo_computational_2011,
title = {Computational design of synthetic regulatory networks from a genetic library to characterize the designability of dynamical behaviors},
author = {Guillermo Rodrigo and Javier Carrera and Alfonso Jaramillo},
doi = {10.1093/nar/gkr616},
year = {2011},
date = {2011-01-01},
journal = {Nucleic Acids Research},
volume = {39},
number = {20},
pages = {e138--e138},
abstract = {The engineering of synthetic gene networks has mostly relied on the assembly of few characterized regulatory elements using rational design principles. It is of outmost importance to analyze the scalability and limits of such a design workflow. To analyze the design capabilities of libraries of regulatory elements, we have developed the first automated design approach that combines such elements to search the genotype space associated to a given phenotypic behavior. Herein, we calculated the designability of dynamical functions obtained from circuits assembled with a given genetic library. By designing circuits working as amplitude filters, pulse counters and oscillators, we could infer new mechanisms for such behaviors. We also highlighted the hierarchical design and the optimization of the interface between devices. We dissected the functional diversity of a constrained library and we found that even such libraries can provide a rich variety of behaviors. We also found that intrinsic noise slightly reduces the designability of digital circuits, but it increases the designability of oscillators. Finally, we analyzed the robust design as a strategy to counteract the evolvability and noise in gene expression of the engineered circuits within a cellular background, obtaining mechanisms for robustness through non-linear negative feedback loops.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}