Analysis of Activity of Human Steroidogenic Acute Regulatory Protein (STARD1) Expressed in Escherichia coli Cells.

One of the main obstacles to the successful use of Escherichia coli cells for steroid transformation in biotechnological processes is inefficient transport of steroid substrates into the cells. Here, we tested the possibility of using human cholesterol transfer protein STARD1 (steroidogenic acute regulatory protein) to increase the efficiency of steroid uptake by bacterial cells. Genetic constructs were obtained for the synthesis in E. coli BL21 (DE3) cells of a truncated version of STARD1 containing protein functional domain (residues 66-285) and STARD1 (66-285)-GFP fusion protein, both carrying bacterial periplasmic targeting sequence pelB at the N-terminus. Analysis of preparations of E. coli/pET22b/STARD1-GFP cells by fluorimetry and Western blotting confirmed that the used expression system ensured the synthesis of the heterologous protein. Using fluorescence spectroscopy, it was demonstrated that the presence of STARD1 in the cells increased the efficiency of assimilation of NBD-labeled cholesterol analogues by E. coli/pET22b/STARD1 cells 1.3-1.6 times (p < 0.05) compared to the wild-type cells, thus demonstrating that human STARD1 exhibits its functional activity in bacterial cells. This opens prospects for optimizing and using a fundamentally new approach to increase the efficiency of steroid uptake by cells - the inclusion of a specific carrier protein in the cell membrane, which can expand the arsenal of methods used to obtain strains of microorganisms for synthesis.

Direct ENIT: An easy and reliable tool for gRNA efficacy verification by tracking induced chromosomal translocation.

CRISPR/Cas systems (Clustered regularly interspaced palindromic repeats / CRISPR-associated) are rapidly becoming a commonplace and popular tool for gene editing in research and clinical contexts. However, the quality of CRISPR/Cas experiments depends heavily on the guide RNA (gRNA) design; therefore, a reliable, easy, and rapid method for verifying gRNA cleavage efficacy is necessary. Engineered nuclease-induced translocations (ENIT) are an easy and cost-efficient method for the verification of gRNA efficacy, which involves tracking induced chromosomal mutations, using polymerase chain reaction (PCR). We have customized this method using both direct PCR and nested PCR approaches and have been able to reduce the sample preparation time. We present a simple and reliable gRNA testing approach that requires no specific enzymes or equipment.•The approach requires only routinely used enzymes and equipment.•Cost- and time-efficient, requiring approximately 30 min for PCR sample preparation, without requiring DNA purification.•High sensitivity, with induced translocation detected in 100 of 10,000 cells in the general population.