HTGAA Class 5a: Cell Free Systems
A cell-free system allows biological reactions to occur outside of living cells. By extracting and using cellular components like ribosomes, RNA polymerase, amino acids, and ATP, this method enables reactions in a controlled, simplified environment. Cell-free systems allow for the engineering, expression, and analysis of genetic constructs without the complexity of living cells.
Applications of Cell-Free Systems include:
Synthetic Biology: Designing and testing biological circuits or pathways without cellular constraints.
Protein Engineering: Rapid protein production and screening, especially for proteins that are toxic or hard to express in cells.
Metabolic Engineering: Production of high-value chemicals, biofuels, and pharmaceuticals via synthetic pathways.
Biosensing: Creating diagnostic tools that are portable and easy to use, like paper-based biosensors.
Gene Editing Research: Testing CRISPR-based systems or genome editing tools in a controlled environment.
In this laboratory, the functionality of the Cell-Free Transcription-Translation (TXTL) system will be demonstrated by expressing the reporter protein amilGFP from a T7-IPTG-inducible plasmid. Unlike traditional systems that use live cells, TXTL relies on biological extracts from them, containing all the machinery and biological components necessary for protein synthesis, combined with a plasmid containing the gene of interest. The primary objective is to quantify protein production by the cell extracts at different IPTG levels to analyze how varying inducer concentrations influence the reaction efficiency across treatments. IPTG serves as the inducer for expression, facilitating the transcription and translation of amilGFP.
The system's activity will be assessed based on amilGFP production, with fluorescence monitored over an 8-hour incubation period. This will be measured using a plate reader or qPCR, with excitation at 492 nm and emission at 506 nm, or as final-point reactions incubated on a thermocycler, ensuring accurate data on protein fluorescence levels. The reaction will be conducted at 30°C, the optimal temperature for maximizing transcription and translation rates while maintaining the stability of enzymatic components in the extract. To halt the reaction, we will reduce the temperature to 4°C, preserving the synthesized protein for further analysis.