Part:BBa_K5101002:Design
Plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 572 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1708
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1678
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
During the design phase, special attention was given to ensure the stability of the AMP gene in the plasmid backbone and the compatibility of the promoter with the E. coli expression system. The RBS was optimized for the E. coli ribosome.
Source
This plasmid, pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7, is constructed using the pET29a vector as the backbone, which is widely used for high-level expression in E. coli. The J23119 promoter, sourced from the iGEM Registry of Standard Biological Parts, is a strong, constitutive promoter that ensures robust expression of downstream genes(BBa_J23119). The promoter SoxR/SoxS, sourced from the iGEM Registry of Standard Biological Parts, is a nitric-oxide-induclble promoter system(BBa_K554003,BBa_K554000). The antimicrobial peptide (AMP) gene included in this plasmid was derived from Lactobacillus spp. from the human vaginal flora(BBa_K5101000). This AMP-expressing Lactobacillus spp. was discovered and initially characterized by our host lab at Nanjing Tech University. The detailed sequence data is pending publication. To align with scientific documentation standards and for comparison, a same sequence of the AMP can be referenced in the NCBI database under Reference Sequence: WP_049159833.1.