Enhanced Golden Gate Assembly: evaluating overhang strength for improved ligation efficiency.

Molecular cloning, a routine yet essential technique, relies heavily on efficient ligation, which can be significantly improved using Golden Gate Assembly (GGA). A key component of GGA is the use of type IIS enzymes, which uniquely cleave downstream of their recognition sequences to generate various overhangs, including non-palindromic ones. Recent advancements in GGA include the development of newly engineered enzymes with enhanced activity.

Robust, high-yield, rapid fabrication of DNA constructs for Magnetic Tweezers.

Single-molecule techniques are highly sensitive tools that can reveal reaction intermediates often obscured in experiments involving large ensembles of molecules. Therefore, they provide unprecedented information on the mechanisms that control biomolecular reactions. Currently, one of the most significant single-molecule assays is Magnetic Tweezers (MT), which probes enzymatic reactions at high spatio-temporal resolutions on tens, if not hundreds, of molecules simultaneously.

Three Microbial Musketeers of the Seas: , and , and Their Adaptation to Different Salinity Probed by a Proteomic Approach.

Osmotic changes are common challenges for marine microorganisms. Bacteria have developed numerous ways of dealing with this stress, including reprogramming of global cellular processes. However, specific molecular adaptation mechanisms to osmotic stress have mainly been investigated in terrestrial model bacteria.

Virus-Host Interaction Gets . PART II: Functional Transcriptomics of the DksA-Deficient Cell upon Phage P1 Infection.

The virus-host interaction requires a complex interplay between the phage strategy of reprogramming the host machinery to produce and release progeny virions, and the host defense against infection. Using RNA sequencing, we investigated the phage-host interaction to resolve the phenomenon of improved lytic development of P1 phage in a DksA-deficient host. Expression of the and P1 genes in the wild-type host was the highest among all and most probably leads to phage virulence.

Virus-Host Interaction Gets . PART I: Phage P1 Enhanced Development in an DksA-Deficient Cell.

Bacteriophage P1 is among the best described bacterial viruses used in molecular biology. Here, we report that deficiency in the host cell DksA protein, an global transcription regulator, improves P1 lytic development. Using genetic and microbiological approaches, we investigated several aspects of P1 biology in an attempt to understand the basis of this phenomenon.

Induction of the Stringent Response Underlies the Antimicrobial Action of Aliphatic Isothiocyanates.

Bacterial resistance to known antibiotics comprises a serious threat to public health. Propagation of multidrug-resistant pathogenic strains is a reason for undertaking a search for new therapeutic strategies, based on newly developed chemical compounds and the agents present in nature. Moreover, antibiotic treatment of infections caused by enterotoxin toxin-bearing strain-enterohemorrhagic (EHEC) is considered hazardous and controversial due to the possibility of induction of bacteriophage-encoded toxin production by the antibiotic-mediated stress.

Adaptation of the Marine Bacterium to Low Temperature Stress.

Marine bacteria display significant versatility in adaptation to variations in the environment and stress conditions, including temperature shifts. plays a major role in denitrification and bioremediation in the marine environment, but is also identified to be responsible for spoilage of ice-stored seafood. We aimed to characterize transcriptional response of to cold stress in order to achieve a better insight into mechanisms governing its adaptation.

Suppression of the Escherichia coli dnaA46 mutation by changes in the activities of the pyruvate-acetate node links DNA replication regulation to central carbon metabolism.

To ensure faithful transmission of genetic material to progeny cells, DNA replication is tightly regulated, mainly at the initiation step. Escherichia coli cells regulate the frequency of initiation according to growth conditions. Results of the classical, as well as the latest studies, suggest that the DNA replication in E.

The Escherichia Coli Hfq Protein: An Unattended DNA-Transactions Regulator.

The Hfq protein was discovered in Escherichia coli as a host factor for bacteriophage Qβ RNA replication. Subsequent studies indicated that Hfq is a pleiotropic regulator of bacterial gene expression. The regulatory role of Hfq is ascribed mainly to its function as an RNA-chaperone, facilitating interactions between bacterial non-coding RNA and its mRNA target.

Hfq protein deficiency in Escherichia coli affects ColE1-like but not λ plasmid DNA replication.

Hfq is a nucleic acid-binding protein involved in controlling several aspects of RNA metabolism. It achieves this regulatory function by modulating the translational activity and stability of different mRNAs, generally via interactions with stress-related small regulatory sRNAs. However, besides its role in the coordination of translation of bacterial mRNA, Hfq is also a nucleoid-associated DNA-binding protein.