Ultra-high throughput mapping of genetic design space.

Massively parallel genetic screens have been used to map sequence-to-function relationships for a variety of genetic elements. However, because these approaches only interrogate short sequences, it remains challenging to perform high throughput (HT) assays on constructs containing combinations of sequence elements arranged across multi-kb length scales. Overcoming this barrier could accelerate synthetic biology; by screening diverse gene circuit designs, "composition-to-function" mappings could be created that reveal genetic part composability rules and enable rapid identification of behavior-optimized variants. Here, we introduce CLASSIC, a generalizable genetic screening platform that combines long- and short-read next-generation sequencing (NGS) modalities to quantitatively assess pooled libraries of DNA constructs of arbitrary length. We show that CLASSIC can measure expression profiles of >10 5 drug-inducible gene circuit designs (ranging from 6-9 kb) in a single experiment in human cells. Using statistical inference and machine learning (ML) approaches, we demonstrate that data obtained with CLASSIC enables predictive modeling of an entire circuit design landscape, offering critical insight into underlying design principles. Our work shows that by expanding the throughput and understanding gained with each design-build-test-learn (DBTL) cycle, CLASSIC dramatically augments the pace and scale of synthetic biology and establishes an experimental basis for data-driven design of complex genetic systems.

Biomaterials-aided mandibular reconstruction using in vivo bioreactors.

Large mandibular defects are clinically challenging to reconstruct due to the complex anatomy of the jaw and the limited availability of appropriate tissue for repair. We envision leveraging current advances in fabrication and biomaterials to create implantable devices that generate bone within the patients themselves suitable for their own specific anatomical pathology. The in vivo bioreactor strategy facilitates the generation of large autologous vascularized bony tissue of customized geometry without the addition of exogenous growth factors or cells. To translate this technology, we investigated its success in reconstructing a mandibular defect of physiologically relevant size in sheep. We fabricated and implanted 3D-printed in vivo bioreactors against rib periosteum and utilized biomaterial-based space maintenance to preserve the native anatomical mandibular structure in the defect site before reconstruction. Nine weeks after bioreactor implantation, the ovine mandibles were repaired with the autologous bony tissue generated from the in vivo bioreactors. We evaluated tissues generated in bioreactors by radiographic, histological, mechanical, and biomolecular assays and repaired mandibles by radiographic and histological assays. Biomaterial-aided mandibular reconstruction was successful in a large superior marginal defect in five of six (83%) sheep. Given that these studies utilized clinically available biomaterials, such as bone cement and ceramic particles, this strategy is designed for rapid human translation to improve outcomes in patients with large mandibular defects.

Effects of Local Antibiotic Delivery from Porous Space Maintainers on Infection Clearance and Induction of an Osteogenic Membrane in an Infected Bone Defect.

Reconstruction of large bone defects can be complicated by the presence of both infection and local antibiotic administration. This can be addressed through a two-stage reconstructive approach, called the Masquelet technique, that involves the generation of an induced osteogenic membrane over a temporary poly(methyl methacrylate) (PMMA) space maintainer, followed by definitive reconstruction after the induced membrane is formed. Given that infection and antibiotic delivery each have independent effects on local tissue response, the objective of this study is to evaluate the interaction between local clindamycin release and bacterial contamination with regards to infection prevention and the restoration of pro-osteogenic gene expression in the induced membrane. Porous PMMA space maintainers with or without clindamycin were implanted in an 8 mm rat femoral defect model with or without Staphylococcus aureus inoculation for 28 days in a full-factorial study design (four groups, n = 8/group). Culture results demonstrated that 8/8 animals in the inoculated/no antibiotic group were infected at 4 weeks, which was significantly reduced to 1/8 animals in the inoculated/antibiotic group. Quantitative polymerase chain reaction analysis demonstrated that clindamycin treatment restores inflammatory cytokine and growth factor expression to the same levels as the no inoculation/no antibiotic group, demonstrating that clindamycin can ameliorate the negative effects of bacterial inoculation and does not itself negatively impact the expression of important cytokines. Main effect analysis shows that bacterial inoculation and clindamycin treatment have independent and interacting effects on the gene expression profile of the induced membrane, further highlighting that antibiotics play an important role in the regeneration of infected defects apart from their antimicrobial properties.