Showing papers on "Lambda phage published in 2022"
TL;DR: In this paper, the authors identify putative TAC encoding genes in diverse T4-like phage genomes and show that the frameshifting mechanism for producing TAC-NC appears to be conserved in several T4 like phage groups.
3 citations
TL;DR: In this paper , the authors identify putative TAC encoding genes in diverse T4-like phage genomes and show that the frameshifting mechanism for producing TAC-NC appears to be conserved in several T4 like phage groups.
3 citations
TL;DR: It is shown that temperature-dependent versions of the Lambda phage repressor CI can serve as tunable cold-actuated transactivators, expanding the toolbox for control of cellular function using globally or locally applied thermal inputs.
Abstract: Temperature is a versatile input signal for the control of engineered cellular functions. Sharp induction of gene expression with heat has been established using bacteria- and phage-derived temperature-sensitive transcriptional repressors with tunable switching temperatures. However, few temperature-sensitive transcriptional activators have been reported that enable direct gene induction with cooling. Such activators would expand the application space for temperature control. In this technical note, we show that temperature-dependent versions of the Lambda phage repressor CI can serve as tunable cold-actuated transactivators. Natively, CI serves as both a repressor and activator of transcription. Previously, thermolabile mutants of CI, known as the TcI family, were used to repress the cognate promoters PR and PL. We hypothesized that TcI mutants can also serve as temperature-sensitive activators of transcription at CI’s natural PRM promoter, creating cold-inducible operons with a tunable response to temperature. Indeed, we demonstrate temperature-responsive activation by two variants of TcI with set points at 35.5 and 38.5 °C in E. coli. In addition, we show that TcI can serve as both an activator and a repressor of different genes in the same genetic circuit, leading to opposite thermal responses. Transcriptional activation by TcI expands the toolbox for control of cellular function using globally or locally applied thermal inputs.
1 citations
TL;DR: This paper cloned the PL promoter from phage lambda into the pRS414 plasmid of over 10kb to create a gene reporter assay, which is very suitable for RNA synthetic biology experiments.
Abstract:
Different gene reporter assays are used for quantifying various methods for synthetic control of gene expression. The activity of a reporter protein, fluorescence or chemical stain, is usually proportional to the level of mRNA produced in the cell and can be utilized for monitoring the activity of a promoter or enhancer. Such a reporter gene is the lacZ from Escherichia coli, encoding β- galactosidase. We cloned the PL promoter from phage lambda into the pRS414 plasmid of over 10kb to create a gene reporter assay. A KpnI restriction site was introduced into the plasmid used for inserting the promoter together with a ribosomal binding site for bacterial and archaeal mRNA and the initiation of transcription codon ATG in frame with the lacZ. B-galactosidase activity assays showed a correlation between the induced expression of the lacZ gene and the time, indicating the rightness of the construct, which can be used for lacZ reporter gene activity assays and synthetic control of gene expression in synthetic biology studies. The new plasmid named pRS414ge is very suitable for RNA synthetic biology experiments.
TL;DR: Since an engineered lambda integrase that is able to efficiently recombine pairs of DNA crossover sites that differ in sequence will be useful for subsequent genome editing in higher eukaryotes to accommodate variations in target site sequences, future inclusion of single cognate sites for other genome targeting systems could enable modularity.
Abstract: Seamless DNA vectors derived from bacterial plasmids are devoid of bacterial genetic elements and represent attractive alternatives for biomedical applications including DNA vaccines. Larger scale production of seamless vectors employs engineered Escherichia coli strains in order to enable tightly regulated expression of site-specific DNA recombinases which precisely delete unwanted sequences from bacterial plasmids. As a novel component of a developing lambda integrase genome editing platform, we describe here strain MG1655-ISC as a means to easily produce different scales of seamless vectors, ranging in size from a few hundred base pairs to more than ten kilo base pairs. Since we employed an engineered lambda integrase that is able to efficiently recombine pairs of DNA crossover sites that differ in sequence, the resulting seamless vectors will be useful for subsequent genome editing in higher eukaryotes to accommodate variations in target site sequences. Future inclusion of single cognate sites for other genome targeting systems could enable modularity. These features, together with the demonstrated simplicity of in vivo seamless vector production, add to their utility in the biomedical space.