Part:BBa_K4297086

PL-CBE-pET-Alloferon-1-eGFP-SD-AmpR

The PL-CBE and the optimized pET plasmid were fused to facilitate the subsequent construction of the RBS library.

Usage and Biology

        In constructing the RBS library and high-throughput screening of pET plasmids, we drew on the NNU-CHINA team and the relevant contents of the literature. We ligated a resistance gene (AmpR) containing a specific SD sequence after the eGFP gene of BBa_K3868102, which can only be produced when the recombinant protein is expressed at high speed. With increasing concentrations of ampicillin, Also, we replaced the RBS sequence of the target gene in the pET plasmid to facilitate the construction of the RBS library using CBE.The obtained pET plasmid library was successfully cultured under high concentration of ampicillin (4000 μg/mL) in accordance with the steps in the design section (Fig 4A). We selected 96 different single colonies for culture and selected three strains with the highest unit fluorescence intensity for further fermentation. The experimental results showed that the unit fluorescence values of the three strains were further improved compared to the original study (up to 5.4-fold) (Fig 4B). Excitingly, the entire screening process takes only 5-7 days and has the potential to be used in conjunction with the RBS library of T7 RNAP.

(A) The workflow of pET plasmid RBS library construction and screening. (B) The unit cell fluorescence intensity of the fermented culture Alloferon-1.