Sensing and Actuation in Miniaturized Systems PRESENTATION DNA

Sensing and Actuation in Miniaturized Systems PRESENTATION DNA Transformation by Local Heat Shock 9533071 陳俊廷

Outline Abstract ¡ Introduction ¡ Design And Fabrication ¡ Experimental Procedure ¡ Results ¡ Conclusions ¡

Abstract ¡ DNA transformation by local heat shock using a MEMS device has been successfully demonstrated using an on-chip micro heater and corresponding microfluidic system ¡ The transformation experiments have been conducted and 200% improvement in transformation efficiency was showed as compared with control tests using conventional methodology

Introduction ¡ Widely used in genetic engineering and molecular biology as an indispensable and key process in gene cloning, gene therapy and protein expression ¡ Among all the DNA transformation methods, electroporation and heat shock are two commonly used procedures. Heat shock devices are easy to make and biocompatible. ¡ Some researchers believe that the abrupt heat pulse makes cell membranes more fluid and permeable for the foreign DNA to transport into cells

1. Bacterial cells, which were treated by chemicals such as Ca. Cl 2 to make the cell membrane permeable 2. Plasmid DNA, a circular DNA that can replicate itself after DNA transformation process

Design and Fabrication ¡ The competent cells and the recombinant plasmids are mixed ¡ The mixture is then treated by a brief heat pulse at 42℃ for 90 seconds for the transformation process ¡ A fluidic cap using molded PDMS to construct micro chamber and channels and a heating substrate with microheaters constructed by resistive heaters with electrical insulation layer on the top

Experimental Procedure ¡ A fine thermocouple (CHAL-0005), was good linearity between 0 to 65℃, TCR of the heater 0. 0015/℃ and the coefficient of determination of the linear fit is 0. 9999

¡ ¡ E. coli stored was thawed on ice and mixed with plasmid DNA, which contains both Venus and ampicillin resistance gene Compare local heat shock, conventional heat shock, and without heat shock

Results ¡ ¡ The micro device achieves 2. 62× 106 colonies/μg, which is about over 200% better than the conventional heat shock result at 1. 13× 106 colonies/μg No transformed cell can be observed without heat shock procedure

Conclusions ¡ A MEMS local heat shock device has been successfully designed, fabricated and tested to demonstrate effective DNA transformation using competent E. coli cells and plasmid DNA with embedded Venus gene. ¡ The improvement in efficiency is likely coming from the effective heating process in the micro scale. As such, this MEMS device has potentials to be part of lab-on-a-chip systems.