The expression and construction of engineering Escherichia coli producing humanized AluY RNAs

Abstract

BackgroundExogenous RNAs can specifically up-regulate or down-regulate gene expression after they enter into cells. Alu RNAs are the main constituent of human transcriptome and participate in gene expression regulation. AluY elements belong to a subfamily of Alus and are the youngest Alus. In this paper, we established the technology method of preparing genetically engineered humanized AluY RNAs (AluY RNAs) from Escherichia coli (E. coli) strains. This technology method also can be used to prepare other genetically engineered humanized RNAs that can be used for cytology experiments.ResultsDifferent copies of human AluY elements were inserted into pET-28α plasmid (pET) to construct pET-AluY plasmids that were transformed into BMBL21-DE3 (DE3) E. coli. Isopropylthio-β-d-galactoside (IPTG) induction inhibited transformed bacterial growth after DE3 E. coli were transformed by pET-AluY × 8 plasmid (8 copies of AluYs were inserted into pET); northern blotting was used to detect the amount of AluY RNAs after 2, 4, 6, 8, 10, 12, 14 and 16 h inducing with IPTG. The results showed that the amount of AluY RNAs was the highest at 4 h; 1, 2, 4, 8 or 14 copies of AluY elements were inserted into the pET to construct pET-AluY plasmids that were transformed into DE3 bacteria, the northern blotting results showed that AluY RNAs production amount increased with the increase of AluY copy number; pET-AluY × 8 DE3 bacteria did not produce AluY RNAs without IPTG induction, AluY RNA production kept similar when inducing by 0.1–0.4 mg/ml IPTG induction, however, AluY RNA production slightly decreased if deviating from the above concentration range; pET-AluY × 8 DE3 bacteria were cultured at 34, 37 or 40 °C and the results showed that AluY RNA production was the highest under 37 °C cultivation; pET-AluY × 8 plasmid was transformed into three kinds of BL21 bacteria, including DE3, BMBL21-DE3-pLysS (pLysS) and Trans BL 21 (TransBL), the results showed that AluY RNA production was the highest when using DE3 bacteria.ConclusionsThe optimal conditions of producing AluY RNAs were: a kind of host bacteria of DE3, an engineering bacteria concentration of OD600 1.0, an IPTG concentration of 0.2 mg/ml, a culturing temperature of 37 °C and a culturing time of 4 h. Pure AluY RNAs occupied 15.8% of extractive total RNAs and the mean yield of pure AluY RNAs in 100 ml bacteria solution was 0.46 mg.

Pharmacological and Toxicological Appraisal of Promising Enzyme-tyrosinase Isolated from Soil Bacteria-an Update

“…Extracellular tyrosinases production in soil environment are most likely involved in detoxification of phenolic compounds of plants, humid matter formation and polymerization [26].…”

Alu antisense RNA ameliorates methylglyoxal-induced human lens epithelial cell apoptosis by enhancing antioxidant defense

“…Cell Culture and Cell Transfection HLECs (SRA01/04, Huatuo, Suzhou, China) were cultured in DMEM medium (4.5 g/L glucose, ThermoFisher Scientific, USA), supplemented with 10% fetal calf serum (FCS; ZETA TM Life, USA) under 5% CO 2 at 37℃. Yeast tRNA (tRNA) was purchased from Solarbio Life Sciences; Alu sense RNA (Alu RNA) and Alu antisense RNAs were prepared by our laboratory [26][27] . These Alu antisense RNAs include AluYas RNA, AluJBas RNA and AluSPas RNA.…”

“…Our previous studies also indicated that murine SINE B1 antisense RNA retards the mouse aging process by removing accumulated ROS in senescent cells and regulating the expression of aging-associated genes [19] . Importantly, there are established methods for producing genetically engineered SINE RNAs that are of sufficient quality for cell and animal experiments [26][27] ; the availability of these methods means that nucleic acid reagents of treating aged-related diseases can be obtained. In this study, we examined whether human SINE antisense RNA (Aluas RNA) can protect human lens epithelial cells (HLECs) from apoptosis induced by MGO and sought to explore the mechanisms by which Aluas RNA could have this effect.…”