We know living systems continuously integrate information from many different sources and that these signals trigger complex and subtle responses. Much of this information processing occurs at the molecular level. We are interested in operating these processes within materials, but without cells.
The purpose is to give materials novel functionality. Challenges in this work are primarily associated with the cell protein synthesis reaction, rather than working with hydrogel. Cell-free reaction require high quality cell lysate as well as high quality and purity DNE to function well.
This protocol allows researchers to embed molecular biology reactions into a physical biodegradable material that can be taken outside of the laboratory. Also, because the reaction output interacts directly with the material chassis, there is potential for the development of materials with novel sensing and response capabilities. In short term, we see these methods as providing a novel route to the deployment of cell-free diagnostic devices.
This protocol allows a number of devices to be created and spatially organized in a manner that is not possible for liquid reactions. Our future interests are twofold. First, we are interested in expanding the nature of the hydrogels we use to bring more material functionality to the work.
Second, we are interested in increasing the complexity and performance of the gene networks we operate in gels, expanding the biological functionality of these systems. To begin, transfer the hydrogels to 1.5 milliliter micro centrifuge tubes, trimming the gels with a scalpel if necessary to make them fit in the wells. In another 1.5 milliliter micro centrifuge tube, combine 10 microliters of the cell-free extract with 25 microliters of 2X cell-free protein synthesis buffer, and four micrograms of plasmid DNA.
Make up to a total volume of 50 microliters with double distilled water to prepare the cell-free protein synthesis solution. Pipette the solution onto the freeze dried hydrogels and allow the gels to soak in the cell-free protein synthesis system for five to 10 minutes at room temperature. Transfer the gels to a black 384 well microtiter plate using a spatula.
Then transfer the microtiter plate to a plate reader and use the plate reader settings shown on the screen for fluorescence detection and analysis. In a 1.5 milliliter micro centrifuge tube on ice, combine 10 microliters of the cell-free extract with four micrograms of plasmoid DNA and 25 microliters of 2X cell-free protein synthesis buffer. Make up to a total volume of 50 microliters with double distilled water.
To prepare the cell-free protein synthesis solution weigh 0.75 grams of agarose, and add it to 100 milliliters of double distilled water buffer to prepare 0.75%agarose. Microwave the 0.75%agarose in 30 second bursts at high power. and pipette 50 microliters of the molten agarose into 1.5 milliliter micro centrifuge tubes, or into molds of a desired shape.
Place the molten agarose on a heat block set to 50 degrees Celsius and allow the agarose to cool but not polymerize. Then mix the molten agarose with a cell-free protein synthesis solution by pipetting and stirring with the pipette tip. Allow the gels to cool to room temperature and polymerize for approximately two minutes.
Transfer the polymerized agarose to 1.5 milliliter micro centrifuge tubes with a spatula and flash freeze in liquid nitrogen. Place the flash frozen hydrogels into minus 80 degrees Celsius storage for one hour. After that, remove the micro centrifuge tube lids.
Cover the tubes with a wax film and pierce the film to allow the moisture to be dried off. Set the temperature of the freeze dryer to minus 20 degrees Celsius and pressure to 0.1 millibars and freeze dry the cell-free protein synthesis devices for 18 hours or overnight. Rehydrate the freeze dried devices for 30 minutes with 50 microliters of double distilled water without excess liquid, and transfer the gels to a black 384 well microtiter plate using a spatula.
Finally, place the microtiter plate on a plate reader and use the plate reader settings shown on the screen for fluorescence detection and analysis. The cell-free protein synthesis of eGFP and mCherry in a hydrogel using E.coli cell lysates is shown in this figure. 0.75%agarose gels were prepared without DNA template with four micrograms of either eGFP or mCherry template or with four micrograms of both eGFP and mCherry template.
An overlay of the two channels is also shown, and the overlay includes the differential interference contrast image. The results confirmed the co-expression of both mCherry and eGFP in agarose.
