SURF 2013: Investigating physical methods of protecting linear DNA fragments in cell-free expression systems
Investigating physical methods of protecting linear DNA fragments in cell-free expression systems
In vitro transcription and translation (TX-TL) reactions are increasingly being used and optimized for protein synthesis and screening purposes. More recently, these systems have become popular for a bottom-up approach to synthetic biology. The Murray Lab uses a cell-free expression toolbox that works with E. coli sigma factors . The E. coli endogenous holoenzyme E7 is used as the primary transcription machinery. Elementary circuit motifs, such as multiple stage cascades, AND gate and negative feedback loops are constructed using gene fragments packed on to a plasmid and have been characterized in this system.
Current protocol requires the use of circuit elements in high concentration as plasmids for optimum transcription and translation. High concentration of plasmids requires ligation of the designed PCR product onto plasmid backbone, transformation into cells and further purification steps.
One way to make the process faster and completely ‘synthetic’ is to use PCR product directly in the cell free reaction mixture. The main advantage of this method is that DNA circuits produced using PCR at high concentration can be directly added to the cell free expression system. However, there are certain exonucleases in the TX-TL system that destroys linear DNA. The aim of this project is to investigate different methods to make PCR products viable in the TX TL system. Several methods including circularizing linear DNA using thermal ligation during PCR, using DNA nanostructures as adaptors  to protect the ends will be investigated during the course of this study.
1. Shin, J., Noireaux, V., An E. coli cell-free expression toolbox: application to synthetic gene circuits and synthetic cell, ACS Synthetic Biology, DOI: 10.1021/sb200016s (2012) 2. Endo M., Miyazaki, R., et al., Transcription regulation system mediated by mechanical operation of a DNA nanostructure, J Am Chem Soc, doi: 10.1021/ja2074856 (2012)