Part:BBa_K2229300:Experience

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Characterization and Improvement by REC-CHENNAI 2022

The upregulation of OmpR234 activates the csgD gene. The csgD gene undergoes transcription followed by translation and finally csgD protein is produced. csgD is the positive transcriptional regulator and it controls the expression of curli proteins csgA, csgB and csgC. Our new composite part BBa_K4509469 has been created to increase the percentage growth in the bacteria. The part BBa_K2229300 submitted by TAS Taipei iGEM 2017 consists of BBa_J23100 promoter. Though the promoter strength of J23100 is high compared to the other promoters, the burden value is J23100 is also high.The burden value of J23100 is 20.0% ± 9.9%. Since the bacteria is producing csgD protein as well as OmpR234 protein, the dual expression of the genes along with the promoter burden should not affect the lifespan of the bacteria. Hence, we decided to reduce the burden value and improve the quality of the bacteria by introducing BBa_J23118 promoter instead of BBa_J23100 into the construct. The burden value of J23118 is only -0.1% ± 3.5%.

Though the assays confirmed that E.coli with BBa_K2229300 led to strong biofilm formation, the percentage reduction in the growth was more for E.coli with BBa_K2229300. From the growth curve graph, it is evident that switching to the constitutive promoter J23118 will reduce the burden value and also helps to enhance the biofilm formation in the E.coli K12 strain. Absorbance was measured at 600 nm.