Effects of downstream genes on synthetic genetic circuits

BACKGROUND: In order to understand and regulate complex genetic networks in living cells, it is important to build simple and well-defined genetic circuits. We designed such circuits using a synthetic biology approach that included mathematical modeling and simulation, with a focus on the effects by which downstream reporter genes are involved in the regulation of synthetic genetic circuits. RESULTS: Our results indicated that downstream genes exert two main effects on genes involved in the regulation of synthetic genetic circuits: (1) competition for regulatory proteins and (2) protein degradation in the cell. CONCLUSIONS: Our findings regarding the effects of downstream genes on regulatory genes and the role of impedance in driving large-scale and complex genetic circuits may facilitate the design of more accurate genetic circuits. This design will have wide applications in future studies of systems and synthetic biology

Construction of a genetic AND gate under a new standard for assembly of genetic parts

<p>Abstract</p> <p>Background</p> <p>Appropriate regulation of respective gene expressions is a bottleneck for the realization of artificial biological systems inside living cells. The modification of several promoter sequences is required to achieve appropriate regulation of the systems. However, a time-consuming process is required for the insertion of an operator, a binding site of a protein for gene expression, to the gene regulatory region of a plasmid. Thus, a standardized method for integrating operator sequences to the regulatory region of a plasmid is required.</p> <p>Results</p> <p>We developed a standardized method for integrating operator sequences to the regulatory region of a plasmid and constructed a synthetic promoter that functions as a genetic AND gate. By standardizing the regulatory region of a plasmid and the operator parts, we established a platform for modular assembly of the operator parts. Moreover, by assembling two different operator parts on the regulatory region, we constructed a regulatory device with an AND gate function.</p> <p>Conclusions</p> <p>We implemented a new standard to assemble operator parts for construction of functional genetic logic gates. The logic gates at the molecular scale have important implications for reprogramming cellular behavior.</p

Multicolor plate reader fluorescence calibration

This is a pre-copyedited, author-produced version of an article accepted for publication in Synthetic Biology following peer review. The version of record Jacob Beal, Cheryl A Telmer, Alejandro Vignoni, Yadira Boada, Geoff S Baldwin, Liam Hallett, Taeyang Lee, Vinoo Selvarajah, Sonja Billerbeck, Bradley Brown, Guo-nan Cai, Liang Cai, Edward Eisenstein, Daisuke Kiga, David Ross, Nina Alperovich, Noah Sprent, Jaclyn Thompson, Eric M Young, Drew Endy, Traci Haddock-Angelli, Multicolor plate reader fluorescence calibration, Synthetic Biology, Volume 7, Issue 1, 2022, ysac010, https://doi.org/10.1093/synbio/ysac010, is available online at: https://doi.org/10.1093/synbio/ysac010.[EN] Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.This work was supported in part by the following funding sources: J.B. was supported by Air Force Research Laboratory (AFRL) and DARPA contract FA8750-17-C-0184. N.S. was supported by funding from the BBSRC under award BB/M011178/1. G.B., L.H., and T.L. were supported by the EPSRC under award EP/R034915/1 and EP/S022856/1. G.C. and L.C. were supported by funds from YF Capital and the National Top Talent Undergraduate Training Program, China. A.V. and Y.B. were funded by Grant MINECO/AEI, EU DPI2017-82896-C2-1-R and MCIN/AEI/10.13039/501100011033 grant number PID2020117271RB-C21. Y.B. was supported by Secretaria de Educaci ' on Superior, Ciencia, Tecnologia e Innovacion-Ecuador (Scholarship Convocatoria Abierta 2011). D.K. was supported by JST, CREST Grant Number JPMJCR21N4, Japan. E.Y. was supported by the National Science Foundation under Grant No. 1939860. J.T. was supported by the Office of the Director of National Intelligence (ODNI), Intelligence Advanced Research Projects Activity (IARPA) under Finding Engineering Linked Indicators (FELIX) program contract N66001-18-C-4507. This document does not contain technology or technical data controlled under either the U.S. International Traffic in Arms Regulations or the U.S. Export Administration Regulations. Views, opinions, and/or findings expressed are those of the author(s) and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government.Beal, J.; Telmer, CA.; Vignoni, A.; Boada-Acosta, YF.; Baldwin, GS.; Hallett, L.; Lee, T.... (2022). Multicolor plate reader fluorescence calibration. Synthetic Biology. 7(1):1-9. https://doi.org/10.1093/synbio/ysac010197

Multicolor plate reader fluorescence calibration

Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells

Comparative analysis of three studies measuring fluorescence from engineered bacterial genetic constructs

Reproducibility is a key challenge of synthetic biology, but the foundation of reproducibility is only as solid as the reference materials it is built upon. Here we focus on the reproducibility of fluorescence measurements from bacteria transformed with engineered genetic constructs. This comparative analysis comprises three large interlaboratory studies using flow cytometry and plate readers, identical genetic constructs, and compatible unit calibration protocols. Across all three studies, we find similarly high precision in the calibrants used for plate readers. We also find that fluorescence measurements agree closely across the flow cytometry results and two years of plate reader results, with an average standard deviation of 1.52-fold, while the third year of plate reader results are consistently shifted by more than an order of magnitude, with an average shift of 28.9-fold. Analyzing possible sources of error indicates this shift is due to incorrect preparation of the fluorescein calibrant. These findings suggest that measuring fluorescence from engineered constructs is highly reproducible, but also that there is a critical need for access to quality controlled fluorescent calibrants for plate readers

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Synthetic Biology and Dual Use

Synthetic biology is science or technology concerning layers of life such as individuals, organs, cells. In this field, components of a layer are combined to construct a system in the upper layer. This paper focuses on studies, at a layer related to gene recombinant experiments, modifying genes and combining multiple genes. By introducing research accomplishments in synthetic biology such as gene networks and synthesis of the whole genome, this paper explains how synthetic biology is an extension of conventional gene engineering and the field of interdisciplinary open innovation. The risks of synthetic biology and risk reduction methods are also introduced.</jats:p