We have engineered synthetic bacteria with genetically designed circuits (GEB). GEBs show intelligent behavior as a consequence of the analogy found between artificial neural networks (ANN) and Boolean logic (Figure A), which regulates the expression of the genetic circuits. First, we have studied different neural networks, i.e. elementary McCulloch-Pitts networks and perceptron, and performed in silico experiments with the gro simulator. Second, using the Cello platform, we obtained the DNA sequence, the logic circuit, and the genetic circuit that emulates the artificial neural network. Third, we edited the plasmid that implements the ANN. The use of this methodology was applied to the treatment of some chronic diseases.
We have developed silk-based hydrogels with different compositions, and analyzed their physical properties. We demonstrated their ability to confine and interact with genetically engineered bacteria at macro- and microscopic scales. Bulk analysis of silk fibroin hydrogels, with and without bacteria, revealed controlled bacterial growth and motility. These findings highlight the potential of silk hydrogels as versatile biomaterials for bacterial encapsulation and controlled microenvironments.
We designed peptides and miniproteins for inhibition of VEGF using a structure-based computational design workflow. The Mini-Z peptide and Z-1-2 protein were used as starting templates and their sequences were redesigned, aiming at improving their VEGF binding affinity and energetic stability. An innovative design pipeline consisting of the artificial intelligence-based tools ProteinMPNN and AlphaFold was established, yielding hundreds of high quality protein designs according to AlphaFold’s confidence metrics. The most promising peptides and proteins were finally selected for wetlab testing. Mini-Z designs were synthesized by solid-phase peptide synthesis and miniproteins were produced by recombinant expression in bacteria.
SPR measurements were established for the determination of binding affinities towards immobilized VEGF. A panel of six different anti-VEGF peptides were tested, establishing the Mini-Z peptide as the best binder with a KD of 13µM. Differences in binding affinity were observed between Mini-Z and its variants with variant M13 being slightly better with a KD of 6.2 µM. In addition, a cell-based bioassay to measure the inhibition of VEGF to the VEGF receptor was established for the functional characterization of anti-VEGF peptides. Amongst the tested peptides Mini-Z showed the strongest inhibition with an IC50 of 16.5 µM.
Engineered recombinant bacteria for survival in silk fibroin hydrogel and anti-VEGF production. Four anti-VEGF peptides were expressed, purified, and confirmed via SDS-PAGE and proteomics. A SLiDE E. coli strain requiring benzothiazole for growth was developed using lambda red recombination. A metabolic cross-feeding system was designed and implemented, optimizing D-alanine production by overexpressing racemase genes and evaluating transporters. A second E. coli strain, auxotrophic for D-alanine, is being engineered with racemase knockouts. A suitable ocular-adapted strain was identified, and a gene expression toolkit is under evaluation for stable gene expression.
