Engineering is a cyclical process, where each attempt brings new insights for the next step.
Aiming to, we designed four experiment cycles under the guidance of DBSL rules. The goals of the four groups of engineering modifications are:
In the first engineering design cycle, our goal is to test whether the engineered bacterial strain selected in the experimental design could grow normally under the experimental conditions.The main objective of this stage is to establish a standardized and highly successful experimental foundation for the subsequent experiments.We have selected the following two strains：DH5-α and Escherichia coli BL21(DE3). DH5α is a mutant strain of Escherichia coli, mainly characterized by its lack of immunity to exogenous DNA. Compared to normal strains, it lacks certain immune mechanisms，can be used to create competent cells for genetic engineering.The Escherichia coli BL21(DE3) strain is a type of Escherichia coli expression strain, using T7 RNA polymerase as the expression system. It can efficiently express genes cloned into expression vectors containing the phage T7 promoter. The λ phage DE3 region contains the T7 phage RNA polymerase gene, and this region is integrated onto the chromosome of BL21.
We used LB solid medium to cultivate DH5-α and Escherichia coli BL21(DE3), observe its growth condition and record. Introduce the plasmid into Escherichia coli BL21(DE3) and DH5-α, then test the transformation process through agarose gel electrophoresis.
By observing its growth rate and colony morphology, we found that this strain can grow normally in the environment we have constructed.This ensured the smooth progress of the subsequent experiments, and through repeated experiments, the influence of the cultivation environment factors on the experimental results was eliminated.
We use a series of protein de novo design tools driven by artificial intelligence, semi-rationalally design the mutation library, who’s prototype bases on MSH-α. Then screened out targeted MSH-α analogues use affinity and biocompatibility as indicators. Finally, the expression systems were introduced into Escherichia coli BL21(DE3) using plasmid pET-28a(+)_Melan-SD.
For more detailed information, please refer to Model.
For more detailed information, please refer to Results.
The short peptides we designed have been proven to have the function of inhibiting melanin production.
Using plasmids, we introduced the TGF-β protein expression system into Escherichia coli, enabling the expression product to simultaneously achieve whitening and anti-aging effects. TGF-β is a type of cytokine that can induce the expression of the c-sis gene in fibroblasts. Define an optimization matrix (media, induction strength, expression temperature; optional promoter/RBS swaps). Pre-register quantitative metrics: live/dead ratio, per-channel intensity, growth metrics.
We used a His-tag enzyme-linked immunosorbent assay (ELISA) to detect the expression of TGF-6xHis, which can indicating the expression and extracellular secretion of TGF-β.
The results showed that compared to the blank control group, E. coli culture supernatant, and BL21 (DE3) culture supernatant, the His signal in the supernatant of the engineered bacteria significantly increased, indicating the successful expression and extracellular secretion of TGF-β. And the supernatants from engineered bacteria expressing only the melanotropin peptide, as well as those co-expressing the melanotropin peptide and TGF, both upregulated collagen expression in L-929 cells. During this cycle, we successfully obtained the dual-effect product as required.
To achieve controlled death of engineered bacteria, we constructed a toxin-antitoxin plasmid regulated by an arabinose operon. Referred to the suicide switch constructed by CAFA_China2022, SZ-SHD, we combined T4 endolysin(Part:BBa_K112806) and T4 holin(Part:BBa_K112805), created a composite suicide switch plasmid. then made adjustments based on BL21(DE3), and other plasmids that were introduced simultaneously. The T4 endolysin is lysozyme from enterobacteria phage T4 degrades peptidoglycan layer. The T4 holin is holins from T4 bacteriophage assemble together to form pores on inner membrane of bacteria allowing lysozyme to reach periplasm and degrade peptidoglycan layer.The combination of the two can mutually enhance each other.
We constructed the pBAD30-T4-lysis plasmid and transformed it into E. coli BL21 (DE3).
We used live/dead bacterial fluorescence staining and colony count-based bactericidal curve measurement to verify whether the suicide switch system can control bacterial apoptosis. The results showed significant death signals in bacteria 24 hours after the addition of arabinose. Colony count results indicated that the number of colonies decreased to zero after 24 hours.
The suicide plasmid can function in E. coli BL21 (DE3) properly.
We designed multiple short peptide sequences using a computer-aided approachand selected three sequences that demonstrated relatively high stability in actualexperiments, which have been submitted as Basic Parts.
They are
BBa_25682NRB,
BBa_25NPAQ79,
and
BBa_257Q5CBQ<
Functional Description: Encoded under the lPTG-inducible T7 promoter, this melanin-inhibitory peptide functions as a competitive peptide analog targeting the a-melanocyte-stimulating hormone receptor (a-MSH). lt effectively blocks melanin synthesis while simultaneously promoting collagen production.