Biology subjects

Jaramillo, A.

Publications and source records attributed to Jaramillo, A..

11 recordsLinked to original sources

bioRxiv2026

Turning of (Ph)age: Expanding the therapeutic potential of our viral allies

Novel strategies for treating bacterial infections are needed to combat the growing threat of antibiotic resistance. Here we engineered P4-like particles for bacterial-culture proof-of-concept experiments in two antimicrobial modes: a single-action design intended to deliver model payloads into bacteria without programmed lysis, and a dual-action design intended to lyse target bacteria while releasing a secondary antigenic payload, exemplified here by model antigens. Using a P2 helper-free P4-like particle production platform, we designed, produced and tested P4-mediated single-and dual-action antimicrobial prototypes. After completing bacterial-culture proof-of-concept experiments, we optimized early-stage bioprocessing for future studies, leading to 1011 plaque forming units (PFU) per mL and 0.25 endotoxin units (EU) per 109 PFU. We also challenged the P4 viral-vector packaging limit by deleting sid to favour packaging into P2-sized capsids ([~]25.8 kb estimated cargo capacity). Importantly, repressing payload expression during particle production improved viral titers by about 2 logs, reduced detected cargo-sequence alterations from 12/20 to 0/20 sequenced post-transduction isolates and enabled higher-dose transduction with stronger detectable antigen signal. Altogether, this study supports P4-derived phage-like particles as antimicrobial prototypes that extend phage function beyond bacterial elimination and provides a basis for future validation in pathogen and animal models of infection.

synthetic biology↗

bioRxiv2026

Engineering bacterial combinatorial promoters for two-input chemical AND switching

Engineering compact bacterial promoters that integrate two chemical inputs would simplify biosensors, synthetic circuits, and dynamic metabolic control, but such promoters remain difficult to build because a second operator can increase partial-state leak, reduce the fully induced state, and create sequence- or reporter-context effects. Here we engineered 12 compact Marionette-derived two-input promoter architectures in Escherichia coli by combining defined template scaffolds with added operators, then quantified 19 reporter-specific four-state truth tables under a predefined operational criterion for chemical-input AND behaviour. Single-input template-promoter controls in matched reporter contexts helped separate weak parent-scaffold output from effects introduced by the second operator. Nine curated architectures passed in at least one reporter, and selected constructs delivered high-utility AND responses, including [11]/max-off separation up to >1,367. The full pass-fail dataset showed that a statistically higher [11] state does not necessarily create a useful switch. Practical utility depended mainly on suppressing [10] and [01] leakage, matching the template scaffold to the inserted operator, and controlling long-operator orientation and local sequence context. Reciprocal architectures behaved differently, and 4 of 7 dual-reporter architectures changed operational classification with reporter context. Thus, this work delivers a compact set of candidate Marionette-compatible two-input promoter parts and a source-traceable design framework for engineering bacterial chemical-input AND switches.

synthetic biology↗

bioRxiv2026

A genetically encoded local learning rule enables physical learning in engineered bacteria

Training physical neural networks directly in matter remains difficult because most platforms do not store and update weights within the same physical substrate. Here we show that engineered Escherichia coli can implement a genetically encoded local learning rule acting on persistent biological memory. We introduce memregulons: bacterial memory elements in which coupled plasmids store an analogue weight as the relative abundance of P1 within the P1+P2 plasmid pool. Environmental inputs activate local promoters, and a shared sublethal kanamycin signal converts promoter activity into differential growth that lowers the stored P1 fraction. In single strains, flow-cytometry trajectories across distinct promoters support the predicted dependence of weight change on learning-channel activity and standing population variance. At the single-cell level, repeated negative learning reshapes the stored weight distribution as it approaches the lower boundary. In mixed populations and co-cultures, one shared negative learning signal selectively rewrites active memregulons, enabling externally routed supervised tic-tac-toe lessons. We then generalise the architecture across orthogonal chemical inputs and combinatorial promoters, and use experimentally measured updates in hybrid analyses of winner-take-all and nonlinear-classifier tasks. These results establish physical learning in living matter through local negative updates of genetically encoded analogue weights.

synthetic biology↗

bioRxiv2023

Accelerated evolution using the phage T7 and its application to evolve riboswitches

Leveraging riboswitches, non-coding mRNA fragments pivotal to gene regulation, poses a challenge in effectively selecting and enriching these functional genetic sensors, which can toggle between ON and OFF states in response to their cognate inducers. Here, we show our engineered phage T7, enabling the evolution of a theophylline riboswitch. We have replaced T7s DNA polymerase with a transcription factor controlled by a theophylline riboswitch and have created two types of host environments to propagate the engineered phage. Both types host an error-prone T7 DNA polymerase regulated by a T7 promoter along with another critical gene - either cmk or pifA, depending on the host type. The cmk gene is necessary for T7 replication and is used in the first host type for selection in the riboswitchs ON state. Conversely, the second host type incorporates the pifA gene, leading to abortive T7 infections and used for selection in the riboswitchs OFF state. This dual-selection system, termed T7AE, was then applied to a library of 65,536 engineered T7 phages, each carrying randomized riboswitch variants. Through successive passage in both host types with and without theophylline, we observed an enrichment of phages encoding functional riboswitches that conferred a fitness advantage to the phage in both hosts. The T7AE technique thereby opens new pathways for the evolution and advancement of gene switches, including non-coding RNA-based switches, setting the stage for significant strides in synthetic biology.

synthetic biology↗

bioRxiv2022

Quantification of genetic variants in bacterial cultures by Sanger sequencing

Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify non-neutral mutations by selection or counter- selection, neutral mutations usually require DNA sequencing to be identified in a population, which are normally expensive and time-consuming. Neutral mutations could even become dominant under changing environmental conditions enforcing transitory selection or counter- selection. We propose a novel methodology to quantify DNA using Sanger sequencing, that we validated experimentally with specially-engineered plasmids both in vitro and in co-transformed E. coli by and assessing our predictions with qPCR and fluorescence quantifications. The method relies on the alignment of the electropherograms from the query and reference samples, where we quantify the DNA concentration from the amplitude ratio of aligned electropherogram peaks. Our DNA quantification will allow quantifying genetic variants, including single-base natural polymorphisms or de novo mutations, from mixed Sanger sequencing reads, with consistent reduction of costs compared to canonical approaches such as qPCR.

synthetic biology↗

bioRxiv2022

Engineered gene circuits with reinforcement learning allow bacteria to master gameplaying

Gene circuits enable cells to make decisions by controlling the expression of genes in reaction to specific environmental factors1. These circuits can be designed to encode logical operations2-7, but implementation of more complex algorithms has proved more challenging. Directed evolution optimizes gene circuits8 without the need for design knowledge9, but adjusting multiple genes and conditions10 in genotype searches poses challenges11. Here we show a multicellular sensor system, AdaptoCells, in Escherichia coli, that can evolve complex behavior through an accelerated adaptation to chemical environments. AdaptoCells recognize chemical patterns and act as a decision-making system. Using an iterative improvement method, we demonstrate that the AdaptoCells can evolve to achieve mastery in the game of tic-tac-toe, demonstrating an unprecedented level of complexity for engineered living cells. We provide an effective and straightforward way to encode complexity in gene circuits, allowing for fast adaptation in response to dynamic environments and leading to optimal decisions.

synthetic biology↗

bioRxiv2021

Gut microbiota of Drosophila subobscura contributes to its heat tolerance but is sensitive to transient thermal stress

Global warming impacts the animals fitness, leading to increasing extinction risk in ectotherm species. Gut microbiota can contribute to host physiology leading to an increase of resistance to abiotic stress conditions. Temperature has profound effects on ectotherms and gut microbiota can influence cold and heat tolerance of ectotherm species. Additionally, the gut microbiota is sensitive to environmental temperature, which induces changes in its composition and diversity. Here, we investigated the role of the gut microbiota on heat tolerance of Drosophila subobscura, comparing the knockdown time between conventional and axenic, and we also assessed the impact of heat stress on the diversity and community structure of the gut microbiota, comparing non-stressed and heat-stressed flies. Our findings provide evidence that gut microbiota influences heat tolerance, and that heat stress modifies the gut microbiota at taxonomical and structural levels. These results demonstrate that gut microbiota contributes to heat tolerance but it is also highly sensitive to transient heat stress, which could have important effects on host fitness, population risk extinction, and vulnerability of ectotherms to current and future climatic conditions.

ecology↗

bioRxiv2020

Comparison of CRISPR and marker based methods for the engineering of phage T7

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 trx 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 fiber 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.

molecular biology↗

bioRxiv2019

Correlated evolution between heat tolerance and thermal performance curves in Drosophila subobscura

Global warming imposes important challenges for ectotherm organisms, which can avoid the negative effects of thermal stress via evolutionary adaptation of their upper thermal limits (CTmax). In this sense, the estimation of CTmax and its evolutionary capacity is crucial to determine the vulnerability of natural populations to climate change. However, these estimates depend on the thermal stress intensity and it is not completely clear whether this thermal stress intensity can impact the evolutionary response of CTmax and thermal reaction norms (i.e. thermal performance curve, TPC). Here we performed an evolutionary experiment by selecting high heat tolerance using acute and chronic thermal stress in Drosophila subobscura. After artificial selection, we found that knockdown temperatures (a CTmax proxy) evolved in selected lines compared to control lines, whereas the realized heritability and evolutionary rate change of heat tolerance did not differ between acute-selected and chronic-selected lines. From TPC analysis, we found acute-selected lines evolved a higher optimal performance temperature (Topt) compared to acute-control lines, whereas this TPC parameter was not different between chronic-selected and chronic-control lines. The evolutionary response of Topt caused a displacement of entire TPC to high temperatures suggesting a shared genetic architecture between heat tolerance and high-temperature performance, which only arose in the acute-selected lines. In conclusion, thermal stress intensity has important effects on the evolution of thermal physiology in ectotherms, indicating that different thermal scenarios conduce to similar evolutionary responses of heat tolerance but do not for thermal performance. Therefore, thermal stress intensity could have important consequences on the estimations of the vulnerability of ectotherms to global warming.

evolutionary biology↗

bioRxiv2019

Engineering the smallest transcription factor: accelerated evolution of a 63-amino acid peptide dual activator-repressor

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 {lambda} is the smallest known repressor (66 amino acids; a.a.) but activators are typically much larger (e.g. {lambda} cI, 237 a.a.). Indeed, previous efforts to engineer a minimal activator from Cro resulted in no activity in vivo. In this study, we show that directed evolution results in a new Cro activator-repressor that functions as efficiently as {lambda} cI, in vivo. To achieve this, we develop Phagemid-Assisted Continuous Evolution: PACEmid. We find that a peptide as small as 63-a.a. functions efficiently as an activator and/or repressor. To our knowledge, this is the smallest protein gene regulator reported to date, highlighting the capacity of transcription factors to evolve from very short peptide sequences.

synthetic biology↗

bioRxiv2018

Quantitative characterization of translational riboregulators using an in vitro transcription-translation system

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 and they have a fast turnover and a low metabolic burden. However, riboregulators are generally designed in silico and tested in vivo, which only provides a yes/no evaluation of their performances, thus hindering the improvement of design algorithms. Here we show that a cell-free transcription-translation (TX-TL) system provides valuable quantitative information about the performances of in silico designed riboregulators. In particular, we use the ribosome as an exquisite molecular machine that detects functional riboregulators, precisely measures their concentration and linearly amplifies the signal by generating a fluorescent protein. We apply this method to 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. 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.

synthetic biology↗