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Comparison of in vivo cell compartmentalisation (left) versus in vitro compartmentalization (right) using double emulsion droplets and fluorescent sorting. 1) In an in vivo system, a library of genes is transformed into bacteria to produce the protein variants and dispersed into a water‐in‐oil (w/o) emulsion aiming to have each droplet contain maximum one cell.
Cell-free protein synthesis, also known as in vitro protein synthesis or CFPS, is the production of protein using biological machinery in a cell-free system, that is, without the use of living cells. The in vitro protein synthesis environment is not constrained by a cell wall or homeostasis conditions necessary to maintain cell viability. [ 1 ]
The T7 promoter region allows large-scale in vitro T7 transcription to transcribe the DNA library into an mRNA library, which provides templates for the in vitro translation reaction later. The ribosomal binding site in the 5’-untranslated region (5’ UTR) is designed according to the in vitro translation system to be
Translation promotes transcription elongation and regulates transcription termination. Functional coupling between transcription and translation is caused by direct physical interactions between the ribosome and RNA polymerase ("expressome complex"), ribosome-dependent changes to nascent mRNA secondary structure which affect RNA polymerase activity (e.g. "attenuation"), and ribosome-dependent ...
The process of transcription is a major source of DNA damage, due to the formation of single-strand DNA intermediates that are vulnerable to damage. [53] The regulation of transcription by processes using base excision repair and/or topoisomerases to cut and remodel the genome also increases the vulnerability of DNA to damage. [53]
Run-off transcription can be used to quantitatively measure the effect of changing promoter regions on in vitro transcription levels, [1] [2] [4] Because of its in vitro nature, however, this assay cannot accurately predict cell-specific gene transcription rates, unlike in vivo assays such as nuclear run-on. [1] [2]
The transcription-translation process description, mentioning only the most basic "elementary" processes, consists of: production of mRNA molecules (including splicing), initiation of these molecules with help of initiation factors (e.g., the initiation can include the circularization step though it is not universally required),
A Riboprobe, abbreviation of RNA probe, is a segment of labelled RNA that can be used to detect a target mRNA or DNA during in situ hybridization. [1] RNA probes can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter.
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