Grasemann, L. | Search bioRxiv

bioRxiv2023

C2CAplus: a one-pot isothermal circle-to-circle DNA amplification system

Rolling circle amplification (RCA) is a widely used DNA amplification method that uses circular template DNA as input and produces multimeric, linear single or double stranded DNA. Circle-to-circle amplification (C2CA) has further expanded this method by implementing product re-circularization using restriction and ligation, leading to a higher amplification yield, and enabling the generation of circular products. However, C2CA is a multistep, non-isothermal method, requiring multiple fluid manipulations and thereby compromises several advantages of RCA. Here, we improved C2CA to implement a one-pot, single step, isothermal reaction at temperatures ranging from 25 to 37{degrees}C. Our C2CAplus method is simple, robust, and produces large quantities of product DNA that can be seen with the naked eye.

synthetic biology↗

bioRxiv2022

Improved cell-free transcription-translation reactions in microfluidic chemostats augmented with hydrogel membranes for continuous small molecule dialysis

Increasing protein production capacity of the PURE cell-free transcription-translation (TX-TL) system will be key to implementing complex synthetic biological circuits, and to establish a fully self-regenerating system as a basis for the development of a synthetic cell. Under steady-state conditions, the protein synthesis capacity of the PURE system is likely at least one order of magnitude too low to express sufficient quantities of all PURE protein components. This is in part due to the fact that protein synthesis cant be sustained during the entire dilution cycle, especially at low dilution rates. We developed a microfluidic chemostat augmented with semi-permeable membranes that combines steady-state reactions and continuous dialysis as a possible solution to enhance protein synthesis at steady-state. In batch operation, the continuous dialysis of low molecular weight components via the membranes extended protein synthesis by over an order of magnitude from 2 hours to over 30 hours, leading to a seven-fold increase in protein yield. In chemostat operation, continuous dialysis enabled sustained protein synthesis during the entire dilution cycle even for low dilution rates, leading to six-fold higher protein levels at steady state. The possibility to combine and independently manipulate continuous dialysis and chemostat operation renders our dialysis chemostat a promising technological basis for complex cell-free synthetic biology applications that require enhanced protein synthesis capacity.

synthetic biology↗