title: Transcription-only cell-free systems and their application in biosensing
Creators: name: Li, Mengxi
Publisher: name: The University of Edinburgh
Year: 2023
Subjects: subject: synthetic biology
subject: cell-free system
subject: biosensors
subject: heavy metals
subject: transcription

description: Living cells are widely used as chassis to carry out various designed functions in traditional synthetic biology, due to their excellent properties in sensing surrounding signals, analysing them and generating responses. By using synthetic biology tools, such as Boolean logic gates, programmable nucleases and genome-encoded cellular memory, we can make the cell specifically recognise the target molecule and respond predictably. These genetically engineered microorganisms have been applied not only in the production of medicines, biofuels and functional biomaterials but also in developing a new generation of biosensors. However, genetically modified organisms raise safety concerns and regulatory issues when applied outside of a laboratory context. Cell-free systems, which inherit the signal recognition and processing functions from donor cells without the issues associated with living organisms, have been developed. Instead of the widely used transcription-translation (TX-TL) cell-free system, a transcription-only (TXO) cell-free system is presented. The TXO system is constructed from three components: cell lysates from chosen microorganisms to provide necessary transcription-related factors, reaction mixture to support the reaction, and DNA circuits to guide signal processing. Firstly, I optimised some conditions of the TXO system. Instead of protein output, the TXO system uses RNA aptamers as output signals. I cross-compared most of the published fluorescent RNA aptamers together with their related fluorophores. Three aptamer-fluorophore complexes with different fluorescence colours, iSpinach-DHFBI (green), Squash-DFHO (yellow) and Mango III (A10U)-YO3-B (red), were selected to use in the TXO reactions. Furthermore, I tested different methods of constructing DNA templates and found that the most efficient way to prepare the templates was overlap extension PCR. Moreover, the supportive reaction buffer, reaction temperature and reaction volume were also optimised. Secondly, another major component of the TXO system, crude cell lysate, was optimised. The growth conditions of the donor microorganism can highly influence the final lysate. Growth conditions were optimized for two donor organisms, Escherichia coli (several strains) and Cupriavidus metallidurans CH34. Moreover, different cell lysis methods, including enzymatic cell lysis, sonication and a combination of lysozyme and sonication were tested. The most effective and reliable system was found to be enzymatic lysis for C. metallidurans CH34 and enzymatic-sonication combination method for E. coli strains. Furthermore, in order to have more target transcription factors (TFs) produced in the lysate, donor microorganisms were modified with additional TF expression genes. Finally, the TXO system has been applied to detect heavy metals in water. Heavy metals in drinking water have long been recognised as a health problem. Some microorganisms, such as E. coli and C. metallidurans, naturally have resistance to some heavy metals. These microorganisms contain TFs that can bind with selected metals and trigger downstream gene expression. Cell lysates from these microorganisms provide necessary transcription-related factors, mainly TFs and RNA polymerase, together with reaction mixture and sensor DNA circuits to make the detection happen. Lysates from C. metallidurans CH34 were found to be capable of sensitive detection of nickel, zinc, lead, mercury, arsenate(III), zinc and copper, although it proved difficult to generate lysates with consistently high activity. Heavy metal detection is not the only application of the TXO system. This system can also be used to detect RNA when combined with the CRISPR system. Other than biosensing, TXO systems can also be used for the screening of TFs and promoters, the selection of RNA polymerases with novel properties, and other important applications. The results presented here should facilitate the use and further development of TXO systems.