Flexible Cellular Platforms for Reconfigurable Biocircuits

Existing methods for cloning and recombination of DNA enable construction of arbitrary sequences. However, the sequential nature of these techniques makes them time-consuming and expensive. Furthermore, while the transformation of an existing plasmid into a host strain can be reliable when a selection marker is used, there are many current limitations: the number of different plasmids that can be co-transformed is limited by the choice of markers and compatible origins of replication; plasmids are less stable than chromosomal DNA and are difficult to maintain indefinitely without mutation; and cistronic interactions cannot be designed since each new nucleotide sequence added is on an unconnected DNA molecule. To overcome these limitations, we are designing reconfigurable chromosomes consisting of both fixed and variable regions. While the fixed region is carefully optimized and tuned ahead of time, the variable region can be modified in the field, at the point-of-use, leading to rapid and on-demand realization of novel biocircuits with many different phenotypes.

The Bio-Field Programmable Gate Array (BioFPGA) and the Bio-Programmable Logic Array (BioPLA) are two variations of reconfigurable biocircuits inspired from their counterparts in digital electronics. The BioFPGA is a chassis that is engineered to allow seamless integration of genes and other nucleotide sequences, while the BioPLA is engineered to enable easy re-wiring of regulatory pathways in existing synthetic circuits. Each chassis is a platform that can support multiple uses and each can be configured differently by an end user to achieve different functions at different times. Both contain one or more configuration bits, or regions of DNA as small as one base pair, that can be mutated to change the functionality of the circuit. The BioFPGA furthermore includes reprogrammable attachment sites (attB, attP, attL and attR sequences, based on the bacteriophage lambda recombination system) and a self-integrating source library plasmid containing multiple useful genes, supporting rapid change and adaptation. Att sites are made reprogrammable by adding an addressing sequence and by engineering mutations that change their specificity. We use recombineering of composite BioBricks to create several new chasses and are gathering results using multiplex automated genome engineering (MAGE) for end-user programming of these reconfigurable biocircuits. Applications include rapid prototyping and characterization, robust circuit design, pharmaceutical screening, and flexible microbial manufacturing.