Advances and Computational Tools towards Predictable Design in Biological Engineering

The design process of complex systems in all the fields of engineering requires a set of quantitatively characterized components and a method to predict the output of systems composed by such elements. This strategy relies on the modularity of the used components or the prediction of their context-dependent behaviour, when parts functioning depends on the specific context. Mathematical models usually support the whole process by guiding the selection of parts and by predicting the output of interconnected systems. Such bottom-up design process cannot be trivially adopted for biological systems engineering, since parts function is hard to predict when components are reused in different contexts. This issue and the intrinsic complexity of living systems limit the capability of synthetic biologists to predict the quantitative behaviour of biological systems. The high potential of synthetic biology strongly depends on the capability of mastering this issue. This review discusses the predictability issues of basic biological parts (promoters, ribosome binding sites, coding sequences, transcriptional terminators, and plasmids) when used to engineer simple and complex gene expression systems in Escherichia coli. A comparison between bottom-up and trial-and-error approaches is performed for all the discussed elements and mathematical models supporting the prediction of parts behaviour are illustrated

Characterization of a synthetic bacterial self-destruction device for programmed cell death and for recombinant proteins release

<p>Abstract</p> <p>Background</p> <p>Bacterial cell lysis is a widely studied mechanism that can be achieved through the intracellular expression of phage native lytic proteins. This mechanism can be exploited for programmed cell death and for gentle cell disruption to release recombinant proteins when <it>in vivo </it>secretion is not feasible. Several genetic parts for cell lysis have been developed and their quantitative characterization is an essential step to enable the engineering of synthetic lytic systems with predictable behavior.</p> <p>Results</p> <p>Here, a BioBrick™ lysis device present in the Registry of Standard Biological Parts has been quantitatively characterized. Its activity has been measured in <it>E. coli </it>by assembling the device under the control of a well characterized N-3-oxohexanoyl-L-homoserine lactone (HSL) -inducible promoter and the transfer function, lysis dynamics, protein release capability and genotypic and phenotypic stability of the device have been evaluated. Finally, its modularity was tested by assembling the device to a different inducible promoter, which can be triggered by heat induction.</p> <p>Conclusions</p> <p>The studied device is suitable for recombinant protein release as 96% of the total amount of the intracellular proteins was successfully released into the medium. Furthermore, it has been shown that the device can be assembled to different input devices to trigger cell lysis in response to a user-defined signal. For this reason, this lysis device can be a useful tool for the rational design and construction of complex synthetic biological systems composed by biological parts with known and well characterized function. Conversely, the onset of mutants makes this device unsuitable for the programmed cell death of a bacterial population.</p

A standard vector for the chromosomal integration and characterization of BioBrick™ parts in Escherichia coli

BACKGROUND: The chromosomal integration of biological parts in the host genome enables the engineering of plasmid-free stable strains with single-copy insertions of the desired gene networks. Although different integrative vectors were proposed, no standard pre-assembled genetic tool is available to carry out this task. Synthetic biology concepts can contribute to the development of standardized and user friendly solutions to easily produce engineered strains and to rapidly characterize the desired genetic parts in single-copy context. RESULTS: In this work we report the design of a novel integrative vector that allows the genomic integration of biological parts compatible with the RFC10, RFC23 and RFC12 BioBrick™ standards in Escherichia coli. It can also be specialized by using BioBrick™ parts to target the desired integration site in the host genome. The usefulness of this vector has been demonstrated by integrating a set of BioBrick™ devices in two different loci of the E. coli chromosome and by characterizing their activity in single-copy. Construct stability has also been evaluated and compared with plasmid-borne solutions. CONCLUSIONS: Physical modularity of biological parts has been successfully applied to construct a ready-to-engineer BioBrick™ vector, suitable for a stable chromosomal insertion of standard parts via the desired recombination method, i.e. the bacteriophage integration mechanism or homologous recombination. In contrast with previously proposed solutions, it is a pre-assembled vector containing properly-placed restriction sites for the direct transfer of various formats of BioBrick™ parts. This vector can facilitate the characterization of parts avoiding copy number artefacts and the construction of antibiotic resistance-free engineered microbes, suitable for industrial use

Non-invasive estimation of intracranial pressure by diffuse optics: a proof-of-concept study

Intracranial pressure (ICP) is an important parameter to monitor in several neuropathologies. However, because current clinically accepted methods are invasive, its monitoring is limited to patients in critical conditions. On the other hand, there are other less critical conditions for which ICP monitoring could still be useful; therefore, there is a need to develop non-invasive methods. We propose a new method to estimate ICP based on the analysis of the non-invasive measurement of pulsatile, microvascular cerebral blood flow with diffuse correlation spectroscopy. This is achieved by training a recurrent neural network using only the cerebral blood flow as the input. The method is validated using a 50% split sample method using the data from a proof-of-concept study. The study involved a population of infants (n = 6) with external hydrocephalus (initially diagnosed as benign enlargement of subarachnoid spaces) as well as a population of adults (n = 6) with traumatic brain injury. The algorithm was applied to each cohort individually to obtain a model and an ICP estimate. In both diverse cohorts, the non-invasive estimation of ICP was achieved with an accuracy of 0.9) and good concordance (Lin's concordance correlation coefficient >0.9) in comparison with standard clinical, invasive ICP monitoring. This preliminary work paves the way for further investigations of this tool for the non-invasive, bedside assessment of ICP.This work leading to the results was funded by the European Union’s Horizon 2020 project “BitMap: Brain injury and trauma monitoring using advanced photonics” (No. 675332); Fundació CELLEX Barcelona; Ministerio de Economía y Competitividad /FEDER (PHOTODEMENTIA, DPI2015-64358-C2-1-R); Instituto de Salud Carlos III / FEDER (MEDPHOTAGE, DTS16/00087 and PI18/00468); the “Severo Ochoa” Programme for Centers of Excellence in R&D (SEV-2015-0522); the Obra social “laCaixa” Foundation (LlumMedBcn); Institució CERCA, AGAUR-Generalitat (2017 SGR 1380); LASERLAB-EUROPE IV; KidsBrainIT (ERA-NET NEURON) and la Fundació La Marató de TV3 (201709.31 and 201724.31).Peer ReviewedPostprint (author's final draft

Curcumin and Novel Synthetic Analogs in Cell-Based Studies of Alzheimer’s Disease

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder that is associated with the most common type of dementia and is characterized by the presence of deposits of the protein fragment amyloid beta (Aβ) in the brain. The natural product mixture of curcuminoids that improves certain defects in innate immune cells of AD patients may selectively enhance Aβ phagocytosis by alteration of gene transcription. In this work, we evaluated the protective effects of curcuminoids in cells from AD patients by investigating the effect on NF-κB and BACE1 signaling pathways. These results were compared to the gene expression profile of the clearance of Aβ. The minor curcumin constituent, bisdemethoxycurcumin (BDC) showed the most potent protective action to decrease levels of NF-κB and BACE1, decrease the inflammatory cascade and diminish Aβ aggregates in cells from AD patients. Moreover, mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase (MGAT3) and vitamin D receptor (VDR) gene mRNAs were up-regulated in peripheral blood mononuclear cells from AD patients treated with BDC. BDC treatment impacts both gene expression including Mannosyl (Beta-1,4-)-Glycoprotein Beta-1,4-N-Acetylglucosaminyltransferase, Vitamin D and Toll like receptor mRNA and Aβ phagocytosis. The observation of down-regulation of BACE1 and NF-κB following administration of BDC to cells from AD patients as a model system may have utility in the treatment of asymptomatic AD patients

Leukocyte Derived Microvesicles as Disease Progression Biomarkers in Slow Progressing Amyotrophic Lateral Sclerosis Patients

The lack of biomarkers in Amyotrophic Lateral Sclerosis (ALS) makes it difficult to determine the stage of the disease in patients and, therefore, it delays therapeutic trials. Microvesicles (MVs) are possible biomarkers implicated in physiological and pathological functions, however, their role in ALS remains unclear. We investigated whether plasma derived microvesicles could be overrepresented in a group of 40 patients affected by ALS compared to 28 Alzheimer’s Disease (AD) patients and 36 healthy volunteers. Leukocyte derived MVs (LMVs) compared to endothelial, platelet, erythrocyte derived MVs, were mostly present in ALS patients compared to AD patients and healthy donors. Correlation analysis corrected for the presence of confounding variables (riluzole, age at onset, site of onset, gender) was tested between PRL (Progression Rate at the Last visit) and LMVs, and a statistically significant value was found (Pearson partial correlation r = 0.407, p = 0.006). We also investigated SOD1, TDP-43 intravesicular protein level in LMVs. Misfolded SOD1 was selectively transported by LMVs and its protein level was associated with the percentage of LMVs in slow progressing patients (r = 0.545, p = 0.033). Our preliminary findings suggest that LMVs are upregulated in ALS patients and they can be considered possible markers of disease progression

Advances and Computational Tools towards Predictable Design in Biological Engineering

The design process of complex systems in all the fields of engineering requires a set of quantitatively characterized components and a method to predict the output of systems composed by such elements. This strategy relies on the modularity of the used components or the prediction of their context-dependent behaviour, when parts functioning depends on the specific context. Mathematical models usually support the whole process by guiding the selection of parts and by predicting the output of interconnected systems. Such bottom-up design process cannot be trivially adopted for biological systems engineering, since parts function is hard to predict when components are reused in different contexts. This issue and the intrinsic complexity of living systems limit the capability of synthetic biologists to predict the quantitative behaviour of biological systems. The high potential of synthetic biology strongly depends on the capability of mastering this issue. This review discusses the predictability issues of basic biological parts (promoters, ribosome binding sites, coding sequences, transcriptional terminators, and plasmids) when used to engineer simple and complex gene expression systems in Escherichia coli. A comparison between bottom-up and trial-and-error approaches is performed for all the discussed elements and mathematical models supporting the prediction of parts behaviour are illustrated