| In vitro: |
| Analytical Biochemistry, 2011, 409(1):130-137. | | Enhancing electro-transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing.[Reference: WebLink] | Bacillus amyloliquefaciens has been a major workhorse for the production of a variety of commercially important enzymes and metabolites for the past decades. Some subspecies of this bacterium are recalcitrant to exogenous DNA, and transformation with plasmid DNA is usually less efficient, thereby limiting the genetic manipulation of the recalcitrant species.
METHODS AND RESULTS:
In this work, a methodology based on electro-transformation has been developed, in which the cells were grown in a semicomplex hypertonic medium, cell walls were weakened by adding glycine (Gly) and DL-Threonine (dl-Thr), and the cell-membrane fluidity was elevated by supplementing Tween 80. After optimization of the cell-loosening recipe by response surface methodology (RSM), the transformation efficiency reached 1.13 ± 0.34 × 107 cfu/μg syngeneic pUB110 DNA in a low conductivity electroporation buffer. Moreover, by temporary heat inactivation of the host restriction enzyme, a transformation efficiency of 8.94 ± 0.77 × 105 cfu/μg DNA was achieved with xenogeneic shuttle plasmids, a 103-fold increase compared to that reported previously. The optimized protocol was also applicable to other recalcitrant B. amyloliquefaciens strains used in this study.
CONCLUSIONS:
This work could shed light on the functional genomics and subsequent strain improvement of the recalcitrant Bacillus, which are difficult to be transformed using conventional methods. | | British Journal of Dermatology, 1984, 111(s27):171-173. | | The effects on wound healing of three amino acids-a comparison of two models[Reference: WebLink] | We have utilized two different models to evaluate the role of the amino acids L-cysteine, glycine and DL-Threonine in wound healing.
METHODS AND RESULTS:
Both studies used a double-blind, placebo-controlled, randomized treatment plan. The first model involved the use of blister bases created on human forearm skin by the ammonium hydroxide technique, whilst the second model was that of hypostatic leg ulceration.
CONCLUSIONS:
We conclude that the amino acids did not contribute to the healing of blister bases but that they were of benefit in hypostatic leg ulceration. |
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