We plan to engineer E.coli BL21(DE3) by introduce gene related to multicopper oxidase domain-containing protein production. Then, we cultivate the engineered bacteria on the UHMWPE membrane using the culture medium conducive to the expression of laccase.
Laccase (multicopper oxidase family protein)
AMC88_RS25540
multicopper oxidase domain-containing protein
AMC88_RS25540
protein coding
Rhizobium phaseoli (strain: R650, biovar: phaseoli, nat-host: Phaseolus vulgaris)
Bacteria; Pseudomonadota; Alphaproteobacteria; Hyphomicrobiales; Rhizobiaceae; Rhizobium/Agrobacterium group; Rhizobium
BL21 (DE3) is a derivative strain of Escherichia coli strain B. E.coli is a commonly used model organism in genetic engineering, with the advantage of whole-genome sequencing, and the gene cloning and expression system is mature and complete. BL21 (DE3) has been further modified and optimized on this basis and is more suitable for the expression of recombinant proteins.Its genotype is F- ompT hsdS (rB-mB-) gal dcm (DE3). Among them, F - indicates that the strain is male sterile and lacks the F plasmid, which helps to reduce plasmid transfer and recombination between cells; the mutation of the ompT gene can reduce the expression level of outer membrane proteases, thereby reducing the hydrolysis of recombinant proteins and increasing the expression yield; the variation of the hsd gene results in the absence of the activity of type I restriction enzymes EcoK or EcoB in the strain cells, improving the transformation efficiency of PCR products; the presence of the dcm gene can eliminate the effect of methylation on adenine and cytosine and help some exogenous plasmids to replicate, such as exogenous plasmids carrying restriction sites that are susceptible to methylation.
T7 RNA polymerase system:
BL21 (DE3) is lysogenized by the λ phage DE3, and the T7 RNA polymerase in phage DE3 is controlled by the lacUV5 promoter. Exogenous addition of IPTG (isopropyl thiogalactoside) can induce the rapid expression of T7 RNA polymerase in the BL21 (DE3) strain, and T7 RNA polymerase then recognizes the T7 promoter on the expression plasmid vector to drive the efficient expression of recombinant proteins.
High expression level:
This strain can highly express heterologous genes, which is very beneficial for some experiments and applications that require large-scale production of recombinant proteins. For example, it has wide applications in the production of some medicinal proteins, industrial enzymes, etc.
Cultivation characteristics:
Fast growth rate: Under suitable cultivation conditions, BL21 (DE3) has a fast growth rate and can reach a high cell density in a short time, which is conducive to increasing the yield of recombinant proteins.
Low cultivation cost:
The cultivation cost of E.coli is relatively low, and the medium composition is simple, without the need for complex nutrients and growth conditions, and it is easy for large-scale cultivation and fermentation.
pET-28a(+)-6xHis
[1] CAO Yuying, WANG Shuang, LIU Yining, et al. Enzymatic characteristics of laccase from Lactobacillus plantarum and its effect on degradation of biogenic amines[J]. Food Science, 2023, 44(12): 157-163. (in Chinese with English abstract)DOI:10.7506/spkx1002-6630-20221025-258. http://www.spkx.net.cn
[2] 孙雨, 张海伦, 王文博, 谭福民, 高翠娟, 刘晶晶. 漆酶的功能特性与微生物生产研究进展[J]. 微生物前沿, 2023, 12(4): 121-129. https://doi.org/10.12677/AMB.2023.124014
[3] ZHU Hong1,2, WANG Hong-wei2, DU Wei2, DAI Chuan-chao2**. Potential role of the endopytic fungus laccase rLACB3 in the bioremediation of peanut continuous cropping soil.[J]. cje.
[4] CAO, Y., WANG, S., LIU, Y., GAO, M., DU, L., MA, L., ZHANG, H., TIAN, X., & YANG, W. (2023). Enzymatic Characteristics of Laccase from Lactobacillus plantarum and Its Effect on Degradation of Biogenic Amines. Food Science, 44(12), 157-163. doi:10.7506/spkx1002-6630-20221025-258