Both Gram-positive and Gram-negative bacteria can take up exogenous DNA when they are in a competent state either naturally or artificially. However, the thick peptidoglycan layer in Gram-positive bacteria's cell wall is considered as a possible barrier to DNA uptake. In the present work, two transformation techniques have been evaluated in assessing the protocol's ability to introduce foreign DNA, pBBRGFP-45 plasmid which harbors kanamycin resistance and green fluorescent protein (GFP) genes into a Gram-positive bacterium, Bacillus cereus EB2. B. cereus EB2 is an endophytic bacterium, isolated from oil palm roots. A Gram-negative bacterium, Pseudomonas aeruginosa EB35 was used as a control sample for both transformation protocols. The cells were made competent using respective chemical treatment to Gram-positive and Gram-negative bacteria, and kanamycin concentration in the selective medium was also optimized. Preliminary findings using qualitative analysis of colony polymerase chain reaction (PCR)-GFP indicated that the putative positive transformants for B. cereus EB2 were acquired using the second transformation protocol. The positive transformants were then verified using molecular techniques such as observation of putative colonies on specific media under UV light, plasmid extraction, and validation analyses, followed by fluorescence microscopy. Conversely, both transformation protocols were relatively effective for introduction of plasmid DNA into P. aeruginosa EB35. Therefore, this finding demonstrated the potential of chemically prepared competent cells and the crucial step of heat-shock in foreign DNA transformation process of Gram-positive bacterium namely B. cereus was required for successful transformation.

中文翻译：

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两种转化方案的评价和阳性质粒导入蜡样芽孢杆菌 EB2 的评估，一种使用定性分析的革兰氏阳性细菌

革兰氏阳性菌和革兰氏阴性菌在天然或人工状态下都可以吸收外源 DNA。然而，革兰氏阳性菌细胞壁中厚厚的肽聚糖层被认为可能是 DNA 摄取的障碍。在目前的工作中，在评估该协议将外源 DNA、带有卡那霉素抗性和绿色荧光蛋白 (GFP) 基因的 pBBRGFP-45 质粒引入革兰氏阳性细菌蜡状芽孢杆菌 EB2 的能力时，对两种转化技术进行了评估。B. cereus EB2 是一种内生细菌，从油棕根中分离出来。革兰氏阴性细菌铜绿假单胞菌 EB35 用作两种转化方案的对照样品。使用各自的化学处理使细胞对革兰氏阳性菌和革兰氏阴性菌具有感受态，并且还优化了选择性培养基中的卡那霉素浓度。使用菌落聚合酶链反应 (PCR)-GFP 定性分析的初步结果表明，蜡状芽孢杆菌 EB2 的假定阳性转化体是使用第二个转化方案获得的。然后使用分子技术验证阳性转化体，例如在紫外光下观察特定培养基上的推定菌落、质粒提取和验证分析，然后进行荧光显微镜检查。相反，两种转化方案对于将质粒 DNA 引入铜绿假单胞菌 EB35 都相对有效。因此，这一发现证明了化学制备的感受态细胞的潜力和革兰氏阳性菌 B. 外源 DNA 转化过程中热休克的关键步骤。