Zhe-Xue Lu

Exploring the mechanism of competence development in Escherichia coli using quantum dots as fluorescent probes.

The mechanism of divalent Ca2+ cation induction of Escherichia coli competence is still not fully understood, though it is a common method for introducing recombinant DNA into bacterial cells in gene engineering. Quantum dots (QDs), as a new fluorescent probe of being applied in biology research, have aroused great interest. In this paper, we explored the mechanism of E. coli competence development using QDs for the first time. Results showed that water-soluble QDs of diameter 3-4 nm could go into competent cells, but could not enter noncompetent cells. This result was further confirmed using atomic force microscopy and DNA transforming experiments, suggesting that nonphysiological, high concentrations of Ca2+ enhanced the penetrability of cell membranes so that QDs, which cannot enter cells normally due to their greater diameter (3-4 nm), can do so easily into competent cells. Therefore, we believe that, at least for E. coli, the mechanism of Ca2+-induced competence development is mediated physicochemically rather than physiologically.

Visualized investigation of yeast transformation induced with Li+ and polyethylene glycol

The effects of Li(+) and polyethylene glycol (PEG) on the genetic transformation of Saccharomyces cerevisiae were investigated by using fluorescence microscopy (FM) to visualize the binding of plasmid DNA labeled with YOYO-1 to the surface of yeast cells, scanning electron microscopy (SEM) and atomic force microscopy (AFM) to image the change in surface topography of yeast cells, coupled with transformation frequency experiments. The results showed that under the same conditions, the transformation frequencies of yeast protoplasts were much higher than those of intact yeast cells. PEG was absolutely required for the binding of DNA to the surface of intact yeast cells or yeast protoplasts, and had no effect on the surface topography of intact yeast cells or yeast protoplasts. In the presence of PEG, Li(+) could greatly enhance the binding of plasmid DNA to the surface of intact yeast cells, increase their transformation frequency, and affect their surface topography. On the other hand, no effect on the DNA binding to the surface of protoplasts and no increase in the number of transformants and no surface topography changes were found upon the treatment with Li(+) to protoplasts. In the present work, the effects of Li(+) and PEG on yeast genetic transformation were directly visualized, rather than those deduced from the results of transformation frequencies. These results indicate that cell wall might be a barrier for the uptake of plasmid DNA. Li(+) could increase the permeability of yeast cell wall, then increase the exposed sites of DNA binding on intact yeast cells. The main role of PEG was to induce DNA binding to cell surface.

Atomic force microscopy in cell biology

The history, characteristic, operation modes and coupling techniques of atomic force microscopy (AFM) are introduced. Then the application in cell biology is reviewed in four aspects: cell immobilization methods, cell imaging, force spectrum study and cell manipulation. And the prospect of AFM application in cell biology is discussed.