SynBioFromLeukipposInstitute

The microbial metabolic versatility found in nature has inspired scientists to create microorganisms capable of producing value-added compounds. Many endeavors have been made to transfer and/or combine pathways, existing or even engineered enzymes with new function to tractable microorganisms to generate new metabolic routes for drug, biofuel, and specialty chemical production. However, the success of these pathways can be impeded by different complications from an inherent failure of the pathway to cell perturbations. Pursuing ways to overcome these shortcomings, a wide variety of strategies have been developed. This chapter will review the computational algorithms and experimental tools used to design efficient metabolic routes, and construct and optimize biochemical pathways to produce chemicals of high interest.

We describe the design of a hybrid system -- a combination of a Dynamic Graphical Model (DGM) with a Deep Neural Network (DNN) -- to identify activities performed during synthetic biology experiments. The purpose is to provide real-time feedback to experimenters, thus helping to reduce human errors and improve experimental reproducibility. The data consists of unlabeled videos of recorded experiments and "weakly supervised" information (i.e., "theoretical" and asynchronous knowledge of sets of high level activity sequences in the experiment) used to train the system. Multiple activity sequences are modeled using a trellis, and deep features are extracted from video images. Model performance is accessed using real-time online statistical inference. The trellis incorporates variations during experiment execution, making our model very general and capable of high performance.

Due to the rich history of Streptomyces as producers of important natural products, this genus of bacteria has recently garnered attention for its potential applications in the broader context of synthetic biology. However, the dearth of genetic tools available to control and monitor protein production precludes rapid and predictable metabolic engineering that is possible in hosts such as Escherichia coli or Saccharomyces cerevisiae. In an effort to improve genetic tools for Streptomyces venezuelae, we developed a suite of standardized, orthogonal integration vectors and an improved method to monitor protein production in this host. These tools were applied to characterize heterologous promoters and various attB chromosomal integration sites. A final study leveraged the characterized toolset to demonstrate their utility to produce the biofuel precursor bisabolene using a chromosomally integrated expression system. These tools advance S. venezuelae to be a practical host for future metabolic engineering efforts.

One of the greatest challenges currently faced in tissue engineering is the incorporation of vascular networks within tissue-engineered constructs. The aim of this study was to develop a technique for producing a perfusable, 3-dimensional, cell-friendly model of vascular structures that could be used to study the factors affecting angiogenesis and vascular biology in engineered systems in more detail. Initially, biodegradable synthetic pseudovascular networks were produced via the combination of robocasting and electrospinning techniques. The internal surfaces of the vascular channels were then recellularized with human dermal microvascular endothelial cells (HDMECs) with and without the presence of human dermal fibroblasts (HDFs) on the outer surface of the scaffold. After 7 days in culture, channels that had been reseeded with HDMECs alone demonstrated irregular cell coverage. However, when using a co-culture of HDMECs inside and HDFs outside the vascular channels, coverage was found to be continuous throughout the internal channel. Using this cell combination, collagen gels loaded with vascular endothelial growth factor were deposited onto the outer surface of the scaffold and cultured for a further 7 days. After this, endothelial cell outgrowth from within the channels into the collagen gel was observed, showing that the engineered vasculature maintains its capacity for angiogenesis. Furthermore, the HDMECs appeared to have formed perfusable tubules within the gel. These results show promising steps towards the development of an in vitro platform for studying angiogenesis and vascular biology in a tissue engineering context.

Chinese Hamster Ovary (CHO) cells are the current industry standard for production of therapeutic monoclonal antibodies at commercial scales. Production optimisation in CHO cells hinges on analytical technologies such as the use of the polymerase chain reaction (PCR) to quantify genetic factors within the CHO genome and to detect the presence of contaminant organisms. PCR-based assays, whilst sensitive and accurate, are limited by (i) requiring lengthy sample preparation and (ii) a lack of standardisation.

Transcription activator-like effectors (TALEs) contain modular programmable DNA binding domains. Fusing TALEs with effector domains creates synthetic transcription factors (TALE-TFs) or nucleases (TALENs), enabling precise gene manipulations. The construction of TALEs remains challenging due to their repetitive sequences. Here we report a simple TALE assembly reaction (STAR) that enables individual laboratories to generate multiple TALEs in a facile manner. STAR uses an isothermal assembly ('Gibson assembly') that is labour- and cost-effective, accessible, rapid and scalable. A small 68-part fragment library is employed, and the specific TALE repeat sequence is generated within ~8 hours. Sequence-verified TALENs or TALE-TF plasmids targeting 17 bp target sequences can be produced within three days, without the need for stepwise intermediate plasmid production. We demonstrate the utility of STAR through production of functional TALE-TFs capable of activating human SOX2 expression. STAR addresses some of the shortcomings of existing Golden Gate or solid-phase assembly protocols and enables routine production of TALE-TFs that will complement emerging CRISPR/Cas9-based reagents across diverse applications in mammalian stem cell and synthetic biology.