Identification of Key Genes during Ca ²⁺ -Induced Genetic Transformation in Escherichia coli by Combining Multi-Omics and Gene Knockout Techniques

Abstract

Using transcriptome and proteome techniques and association analysis, we identified several key genes involved in the formation of Ca 2+ -mediated E. coli DH5α competent cells. We used Red homologous recombination technology to construct three single-gene deletion strains and found that the transformation efficiencies of yiaW , ygiZ , and osmB deletion strains for different-size plasmids were significantly increased.

Bioinformatics analysis of gene bhsA and its role in Ca2+‐treated Escherichia coli

“…In contrast, sterile water was used in place of 100 mmol/L CaCl 2 as a negative control. After a successful transformation, the transformation efficiency of each plasmid was calculated using the following formula [16]: $$$\mathrm{Transformation}\unicode{x02007}\mathrm{efficiency}\unicode{x02007}(\mathrm{CFU}/\mathrm{\mu g})=\frac{D\times C\times Tv}{Bv\times Q},$$$where D is dilution times; C is plate colony number; Tv is volume of conversion solution (µL); Bv is volume of coating bacterial solution (µL); Q is plasmid quality number (ng).…”

“…To prepare the samples for scanning electron microscopy (SEM), the cells were dehydrated using gradient concentrations (30%, 50%, 70%, 80%, 90%) of ethanol solution and finally ethanol was replaced with tert ‐butanol three times before drying at room temperature. The dried films were plated and observed by SEM [16].…”

“…In contrast, sterile water was used in place of 100 mmol/L CaCl 2 as a negative control. After a successful transformation, the transformation efficiency of each plasmid was calculated using the following formula [16]:…”

“…In our previous work [16], transcriptomics and proteomics methods were applied to study the changes in transcription and translation levels in E. coli DH5α treated with Ca 2+ to form a sensing state. Through correlation analysis, 24 major genes, including yiaW , loiP , and bhsA , were found that may play key roles in competent cells preparation.…”