Synthetic minimal cells are a class of small liposome bioreactors that have some, but not all fun... more Synthetic minimal cells are a class of small liposome bioreactors that have some, but not all functions of live cells. Here, we report a critical step towards the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes, and transport of nucleic acid payloads by RNA binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of...
Efficient cell-free protein expression from linear DNA templates has remained a challenge primari... more Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. Here we present a modified T7 RNA polymerase promoter that acts to significantly increase the yields of both transcription and translation within in vitro systems. The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system. Unlike other methods of limiting linear template degradation, the T7Max promoter increases transcript concentration in a T7 transcription reaction, providing more mRNA for translation.
Synthetic minimal cells are a class of small liposome bioreactors that have some, but not all fun... more Synthetic minimal cells are a class of small liposome bioreactors that have some, but not all functions of live cells. Here, we report a critical step towards the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes, and transport of nucleic acid payloads by RNA binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of...
Efficient cell-free protein expression from linear DNA templates has remained a challenge primari... more Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. Here we present a modified T7 RNA polymerase promoter that acts to significantly increase the yields of both transcription and translation within in vitro systems. The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system. Unlike other methods of limiting linear template degradation, the T7Max promoter increases transcript concentration in a T7 transcription reaction, providing more mRNA for translation.
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Papers by Kaitlin Stokes