Multilevel Regulation and Translational Switches in Synthetic Biology

In contrast to the versatility of regulatory mechanisms in natural systems, synthetic genetic circuits have been so far predominantly composed of transcriptionally regulated modules.Thisisabouttochangeastherepertoireoffoundational tools for post-transcriptional regulation is quickly expanding. We provide an overview of the different types of translational regulators: protein, small molecule and ribonucleic acid (RNA) responsive and we describe the new emerging circuit designs utilizing these tools. There are several advantages of achieving multilevel regulation via translational switches and it is likely that such designs will have the greatest and earliest impact in mammalian synthetic biology for regenerative medicine and gene therapy applications

Cell-free protein synthesis system as a prototyping platform for mammalian synthetic biology

As the field of Synthetic Biology is expanding, there is an increasing need for methods to develop new modes of synthetic gene regulation. A demand for measurement methodologies which provide results that can be interpreted outside a specific context and used to design larger composite devices in diverse cell types is especially urgent. The motivation of my PhD research was to establish new prototyping tools to measure gene regulation, specifically for mammalian applications. Two different experimental approaches were explored. At first, a tissue culture based method was adapted to measure gene expression regulation. However, it was found that this method would not scale to testing many variants, and alternatives needed to be sought. The results from characterising an RNA binding protein which can repress translation (L7Ae) in cells were used as a reference point to compare later obtained results using the second cell-free measurement approach. The hypothesis of this thesis comprises of establishing a new cell-free protein synthesis (CFPS) method as a prototyping tool for mammalian gene expression. The sections involve an initial proof-of-concept phase, the development of a semi-automated workflow, discovery of novel findings using the method, and preliminary work with the aim of establishing a mathematical modelling framework. The hypothesis that CFPS is an appropriate model for gene expression in mammalian cells is proven across the biochemical analysis of transcription and translation steps. The potential of CFPS was demonstrated through prototyping diverse modes of gene regulation, including transcription and translation regulation. Specifically, T7 constitutive promoter variants, IRES constitutive translation-initiation sequence variants, CRISPR/dCas9-mediated transcription repression, and L7Ae-mediated translation repression were characterised. Liquid handling technology was used to automate reaction assembly and software tools were developed to aid the experimental process throughout design, implementation and data analysis. A graphical user interface was designed to enable wide use of the method. The robustness of the method was established through comparison of repeated experiments. In the last section, the feasibility to measure RNA production in real time in CFPS was established. A mathematical model of bacterial CFPS was adapted to the mammalian context, and preliminary parameter inference was carried out, based on experimental data. Overall, the results contribute to the understanding of the biochemistry of L7Ae, CRISPR/Cas9 and transcription in mammalian systems. The established automated methods will impact mammalian synthetic biology by enabling further work at a faster scale than would be possible using traditional experimental approaches.Open Acces

P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>.

<p><i>E</i>. <i>coli</i> MG1655 transformed with empty vector, native, constitutive or hybrid <i>phaCAB</i> constructs were cultured in 1 liter LB media, supplemented with 3% glucose (w/v) for 24 hours or 48 hours. P(3HB) was purified from these cultures and measured as <b>(A)</b> P(3HB) production (g/L) and <b>(B)</b> P(3HB) content (weight [wt.] % of cell dry weight [CDW]). Data represent the mean +/- the standard deviation of three independent experiments. Student t-test, *P<0.05, **P <0.01, ***P <0.001 and ****P <0.0001.</p

Flow cytometry analysis of P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i> from waste-media cultures.

<p><i>E</i>. <i>coli</i> MG1655 transformed with either empty vector [EV], native [N], constitutive [C] or hybrid [H] <i>phaCAB</i> constructs were cultured in 5 ml of waste-media for 36 h at 37°C. P(3HB) content was assessed via flow cytometry analysis of Nile Red staining. (<b>A</b>) Representative forward scatter (FSC) and side scatter (SSC) contour plots. (<b>B</b>) Representative histogram (FL-5). (<b>C</b>) Normalized fluorescence of Nile Red stained <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>, from three independent experiments. Error bars, +/- the standard deviation. Student t-test, *P<0.05 and ***P <0.001.</p

<i>phaCAB</i> pathway and constructs.

<p>(<b>A</b>) Schematic of poly-3-hydroxybutyrate (P(3HB)) production via the <i>phaCAB</i> operon pathway. (<b>B</b>) The constructs used in this study. Abbreviations: Pwt (wildtype promoter; green arrow), J23104 (Anderson constitutive promoter, BBa_J23104; red arrow), B0034 (ribosomal binding site, BBa_B0034; red half-circle), <i>phaC</i> (PHA synthase), <i>phaA</i> (3-ketothiolase) and <i>phaB</i> (acetoacetyl-CoA reductase). Green half-circles denote native ribosomal binding sites. Construct symbols are based on the Synthetic Biology Open Language Visual (SBOLv) v1.0.0 guidelines [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117202#pone.0117202.ref021" target="_blank">21</a>].</p

Simulated P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>.

<p>In order to simulate P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>, a P(3HB) synthesis model was constructed using the Simbiology toolbox of Matlab. Using this model the flux of several metabolites and species were simulated in order to identify aspects of the system that could be selectively tuned to increase the production of P(3HB). From these analyses, several novel <i>phaCAB</i> operons were designed. These data show the simulated P(3HB) production across several different <i>phaCAB</i> operon designs, where <i>phaCAB</i> expression is under the control of the indicated Anderson constitutive promoters.</p