Endoribonucleases are enzymes that cleave RNA molecules within their sequence. Recent studies have shown that these enzymes are important in the regulation of mRNA. There are specific endoribonucleases for different types of mRNA that need to be degraded. For example, cytoplasmic mRNA is degraded in a different way than functional mRNA. This is a mechanism to decrease protein levels since it degrades the mRNA from which a peptide is translated. Without the template to create new peptides, there will not be more protein production. Furthermore, the existing proteins can be degraded by other mechanisms, like proteases.
Proteases are enzymes that degrade peptide bonds through hydrolisis, causing the degradation of proteins. This enzymes are key in the control of protein levels within the cell. One difference between proteases and endoribonucleases is the way they identify their target when regulating protein levels within a cell. Proteases target peptide chains by tags added to the protein by other enzymes, like the addition of ubiquitin. ERNs recognize the mRNA by specific sequences within the nucleotide chain. Another difference between the mechanisms is the type of break they make in the molecule they are degrading. Proteases phosphorilyze the peptidic bonds between amino acids in order to degrade the protein. ERNs hydrolize the phosphodiester bonds that join the nucleotides together in RNA.
https://www.embopress.org/doi/full/10.1093/emboj/19.1.94
https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2009.07488.x
https://www-sciencedirect-com.ezbiblio.usfq.edu.ec/science/article/pii/B9780128161098000209
Lipofectamine 3000 is a transfection reagent of high efficiency and low impact on the cell. This reagent helps with lipid-based transfection. The DNA is transfected into the cell by being encased in a liposome. Lipids have a positive charge, while the cell membrane and DNA have negative charges. By enclosing the DNA in a positive vessle (liposome), it can enter the cell through endocytosis by the interaction of the charges of these molecules. Endocytosis is the process through which an endosome forms around the liposome (cell membrane surrounds it) and it is released within the cell. Usually, the endosome matures inside the cell and becomes a lysosome, which will degrade the contents within the endosome. However, Lipofectamine 3000 supresses the maturation, releasing the DNA from the endosome and helping its transit to the nucleus of the cell.
https://theory.labster.com/mechanism-lipofectamine-3000/
Poly-transfection is the process by which multiple genetic constructs can be introduced into a cell in one reaction. Multiple copies of DNA can be introduced, which makes it so that all the cells in a population recieve different ratios of the genes or constructs. This facilitates the process of figuring out the stochiometry of the different proteins, transcription factors and molecules needed to make a circuit work.
Poly-transfection is especially useful when working with neuromorphic circuits because of their complexity. Various genes need to be expressed in order for neuromorphic circuits to work properly. By expressing them in different ratios, researchers can figure out the appropriate concentrations of the genes that will make a circuit express the desired function.
https://pubmed.ncbi.nlm.nih.gov/36912522/
https://www.frontiersin.org/journals/neuroscience/articles/10.3389/fnins.2011.00073/full
Genetic toggle switches were first described by Gardner, Gantor & Collins in their article Construction of a genetic toggle switch in Escherichia coli published in 2000. It consists of a synthetic, bistable gene-regulatory network. It uses two repressible promoters that are mutually inhibitory, so they can regulate themselves and achieve a bi-stability.
To create a genetic toggle switch you first need two repressible promoters. The genes that they express need to include the repressor for the other promoter. The promoters also need to be inducible, so that when there is a transcient external stimuli added to the medium where the cells are growing, like a chemical or heat, it can activate the promoter that is not being currently expressed. This stimuli helps the system make the switch to the other pathway. The promoter in the other pathway expresses the repressor for the first pathway, making it so that only one is being expressed at a time.