Abstract
Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.
Highlights
RNA with substantial structure, such as double-stranded RNA, is a poor template for protein synthesis (Lee et al 1993; Fire et al 1998; Hamilton and Baulcombe 1999; Elbashir et al 2001)
To investigate whether bacterial penetron was utilized to gene silencing technique, we introduced artificially synthesized antisense oligonucleotide DNA into bacterial cells by using sliding friction and sepiolite
Introduction of antisense oligonucleotide DNA into E. coli penetrons and evaluation of gene silencing effect on β-lactamase A 50 μl aliquot of sepiolite solution containing 0.2 μM of antisense oligonucleotide DNA (B-LAC15, B-LAC90), 200 mM NaCl, and an equivalent volume of E. coli culture broth were streaked onto the surface of 2% agar hydrogel containing LB contents and 100 μg/ml of ampicillin
Summary
RNA with substantial structure, such as double-stranded RNA, is a poor template for protein synthesis (Lee et al 1993; Fire et al 1998; Hamilton and Baulcombe 1999; Elbashir et al 2001). E. coli penetrons and evaluation of gene silencing effect on β-lactamase A 50 μl aliquot of sepiolite solution containing 0.2 μM of antisense oligonucleotide DNA (B-LAC15, B-LAC90), 200 mM NaCl, and an equivalent volume of E. coli culture broth were streaked onto the surface of 2% agar hydrogel containing LB contents and 100 μg/ml of ampicillin. SDS-PAGE and analysis of protein bands A 100 μl mixture containing 50 μl of sepiolite solution with 0.2 μM of antisense DNA oligonucleotide (LacZ15, LacZ90), 200 mM NaCl and 50 μl of E. coli culture broth was streaked onto the surface of a 2% agar hydrogel containing 1 mM IPTG and 200 mM NaCl. Again, each mixture was exposed to the sliding friction field between a streak bar and agar hydrogel for 60 sec.
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