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#6930. Streamlining cell-free protein synthesis biosensors for use in human fluids: In situ RNase inhibitor production during extract preparation
| December 2026 | publication date |
| Proposal available till | 01-06-2025 |
| 4 total number of authors per manuscript | 0 $ |
| The title of the journal is available only for the authors who have already paid for |
| Journal’s subject area: |
| Environmental Engineering; Biotechnology; Biomedical Engineering; Bioengineering; |
| place 1 | place 2 | place 3 | place 4 |
| Free | Free | Free | Free |
| 2350 $ | 1200 $ | 1050 $ | 900 $ |
Contract №6930.1   | Contract №6930.2 | Contract №6930.3 | Contract №6930.4 |
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Abstract:
Cell-free protein synthesis (CFPS) biosensors provide flexible, fast, and on-demand diagnosis of biomarkers in human body fluids. Considering the high concentrations of many different RNases in human body fluids, RNase inhibitor is an important reagent for the human-body-fluid-based biosensors and accounts for more than 90% of the total cost of E. coli-lysate-based CFPS biosensors. Here an E. coli extract with RNase inhibition activity was developed which reduces the total cost of a CFPS biosensor by ~90%. Murine RNase inhibitor (m-RI) was overexpressed in E. coli cells at different growth temperatures, redox buffers, and folding chaperones. Reduced growth temperature accompanied by overexpression of GroEL/ES folding chaperones were found to be necessary to maintain RNase inhibition activity despite reduced m-RI expression capability. Further, to optimize protein synthesis capability of CFPS biosensors, E. coli extract containing m-RI was mixed with regular E. coli extract that did not contain m-RI. The optimized CFPS biosensor reagent mixture was lyophilized and rehydrated with 0–100% (v/v) of three human body fluids of saliva, serum, and urine. Rehydration with saliva showed the least drop in protein expression compared to serum and urine. Finally, the CFPS biosensor reagents were assessed with a saliva-based CFPS biosensor sensitive to glutamine concentrations.
Keywords:
Biosensors; Cell-free protein synthesis; CFPS; E. coli extract; Human body fluids; RNase inhibitor
Contacts :
2 place - free (for sale)
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Abstract:
Cell-free protein synthesis (CFPS) biosensors provide flexible, fast, and on-demand diagnosis of biomarkers in human body fluids. Considering the high concentrations of many different RNases in human body fluids, RNase inhibitor is an important reagent for the human-body-fluid-based biosensors and accounts for more than 90% of the total cost of E. coli-lysate-based CFPS biosensors. Here an E. coli extract with RNase inhibition activity was developed which reduces the total cost of a CFPS biosensor by ~90%. Murine RNase inhibitor (m-RI) was overexpressed in E. coli cells at different growth temperatures, redox buffers, and folding chaperones. Reduced growth temperature accompanied by overexpression of GroEL/ES folding chaperones were found to be necessary to maintain RNase inhibition activity despite reduced m-RI expression capability. Further, to optimize protein synthesis capability of CFPS biosensors, E. coli extract containing m-RI was mixed with regular E. coli extract that did not contain m-RI. The optimized CFPS biosensor reagent mixture was lyophilized and rehydrated with 0–100% (v/v) of three human body fluids of saliva, serum, and urine. Rehydration with saliva showed the least drop in protein expression compared to serum and urine. Finally, the CFPS biosensor reagents were assessed with a saliva-based CFPS biosensor sensitive to glutamine concentrations.
Keywords:
Biosensors; Cell-free protein synthesis; CFPS; E. coli extract; Human body fluids; RNase inhibitor
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