Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities

Construction of 5-Aminolevulinic Acid Microbial Cell Factories through Identification of Novel Synthases and Metabolic Pathway Screens and Transporters

“…However, previous studies have mainly focused on the regulation of central carbon metabolic pathways ostensibly related to 5-ALA synthesis, such as hemB , odhI , and sdhA . In order to identify metabolic genes that have a less direct connection to 5-ALA synthesis, we constructed a de novo designed ssl -sRNA (single stem-loop sRNA) library targeting 101 potential metabolic targets in central carbon metabolism and amino acid metabolism pathways and examined 5-ALA production after repressing the depicted genes (Figure A). This library allows us to semirationally identify new metabolic targets inhibiting 5-ALA synthesis.…”

“…We devised a 21 nt sequence to bridge the ATG and 5′UTR regions of different potential target genes, creating synthetic single stem-loop sRNAs ( ssl -sRNAs) with inhibitory capabilities as described . Subsequently, we integrated the 24 nt reverse-paired bases, stem-loop section, and PolyU in a one-pot PCR approach to construct the intended inhibitory sRNA as described previously .…”

“…We devised a 21 nt sequence to bridge the ATG and 5′UTR regions of different potential target genes, creating synthetic single stem-loop sRNAs (ssl-sRNAs) with inhibitory capabilities as described. 26 Subsequently, we integrated the 24 nt reverse-paired bases, stem-loop section, and PolyU in a one-pot PCR approach to construct the intended inhibitory sRNA as described previously. 26 To identify the 21 nt gene sequence corresponding to the potential target gene in the genome of E. coli BL21 (DE3), we utilized the ssl-sRNA scoring function and accessed the web server sslRNAD (http://www.kangzlab.cn/).…”

“…26 Subsequently, we integrated the 24 nt reverse-paired bases, stem-loop section, and PolyU in a one-pot PCR approach to construct the intended inhibitory sRNA as described previously. 26 To identify the 21 nt gene sequence corresponding to the potential target gene in the genome of E. coli BL21 (DE3), we utilized the ssl-sRNA scoring function and accessed the web server sslRNAD (http://www.kangzlab.cn/). This automated procedure facilitated the generation of the sRNA sequence, along with the corresponding construction primers.…”

“…Figure 2.Global screening of target genes for the promotion of 5-ALA synthesis using an ssl-sRNA library. (A) Generation of ssl-sRNA libraries targeting 101 depicted genes with one-pot PCR with 101 pairs of primer mixture as described in our previous publications 26. The metabolic pathway for 5-ALA biosynthesis from the glucose via the C4 pathway is highlighted with a purple line, amino acids are indicated with a lightcolored box, and target genes for rescreening are marked with a gray dot.…”