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Construction and application of membraneless organelles in prokaryotes

An organelle and concatenated technology, which is applied to the construction and application of membraneless organelles in prokaryotes, can solve the problems of hindering the exchange of materials in compartments and increase the complexity of artificial synthesis, and achieve the effect of retaining biological activity and rapid material exchange.

Active Publication Date: 2021-03-30
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The main compartmentalization strategy in the prior art in prokaryotic cells is to overexpress multiple bacterial microcompartment-associated proteins to imitate natural bacterial microcompartments. Participation increases the complexity of artificial synthesis; there is a physical barrier composed of proteins between the formed compartment and the cytoplasm, which hinders the exchange of substances inside and outside the compartment

Method used

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  • Construction and application of membraneless organelles in prokaryotes
  • Construction and application of membraneless organelles in prokaryotes
  • Construction and application of membraneless organelles in prokaryotes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Induces Escherichia coli to form membraneless compartments

[0032] The specific steps of this example include: constructing a protein expression vector pET28a4-MaspI16 composed of 16 repeat peptide monomers of spider dragline protein MaSp1 (amino acid composition is GRGGLGGQGAGAAAAAGGAGQGGYGGLGSQG), and transforming it into the expression host cell Escherichia coli BL21 In (DE3), the recombinant expression strain was cultured overnight at 37°C / 220rpm in 4 mL LB medium containing kanamycin (0.05mg / mL), and transferred to 20mL medium containing kanamycin at an inoculum size of 1%. In LB medium, culture at 37°C / 220rpm until OD600 is about 0.6, add 200μM IPTG, continue to culture for 6h, take samples, and verify the solubility of the target protein by SDS-PAGE, such as figure 1 ; Escherichia coli intracellular compartments were visualized under a microscope by Tht staining, as figure 2 ; Observation of E. coli intracellular compartments by transmission electron microscop...

Embodiment 2

[0039] Construction of fluorescently active membrane-free compartments

[0040] The specific steps of this example include: construction of 16 recombinant spidroin protein Masp1 repeat peptide concatenations and GFPmut protein fusion protein expression vector pET28a4-MaspI16-gfp; the GFPmut peptide of the fusion protein is connected as shown in Seq ID No.4 The peptide (the amino acid composition is GGSGGSGGSGGS) is connected to the C-terminus of the peptide concatenation.

[0041] The above expression vector was transformed into the expression host cell Escherichia coli BL21 (DE3), and the recombinant expression strain was cultured overnight at 37° C. / 220 rpm in 4 mL LB medium containing kanamycin (0.05 mg / mL) to Transfer 1% of the inoculum into 20mL LB medium containing kanamycin, cultivate at 37℃ / 220rpm until the OD600 is about 0.6, add 200μM IPTG, continue to cultivate for 6h, take samples, and observe the intracellular region of Escherichia coli under a fluorescent microsc...

Embodiment 3

[0044] Colocalization of two fluorescent proteins in the membrane-free compartment

[0045]The specific steps of this example include: constructing 16 recombinant spidroin protein Masp1 repeat peptide concatenations and GFPmut protein fusion protein expression vector PET28a4-MaspI16-gfp; constructing 16 recombinant spidroin protein Masp1 repeat peptide concatenations fused with mcherry protein Protein expression vector PACYC-MaspI16-rfp; construction of 16 recombinant spidroin protein Masp2 repeat peptide concatenations and mcherry protein fusion protein expression vector PACYC-MaspII16-rfp; construction of 32 arthropod elastin-like conserved peptide concatenations and mcherry protein Fusion protein expression vector PACYC-R32-rfp

[0046] Transform any expression vector of pET28a4-MaspI16-gfp and pACYC-MaspI16-rfp / pACYC-MaspII16-rfp / pACYC-R32-rfp into the expression host cell Escherichia coliBL21 (DE3), and the recombinant expression strain Cultivate overnight at 37°C / 220rpm...

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Abstract

The invention discloses construction and application of a membraneless organelle in prokaryotic escherichia coli. According to the method, an expression vector of recombinant spider silk protein or arthropod-like elastin of protein consisting of a tandem mass of repeated peptide segment monomers of nephila cavaleriei dragline silk protein MaSp1 shown as Seq ID No. 1 and protein consisting of a tandem mass of MaSp2 repeated peptide segment monomers shown as Seq ID No. 2 is constrcuted, and is introduced into an expression host; after induced expression, a soluble protein enrichment phase, namely a membrane-free chamber, is formed; and component protein in the membrane-free area exists in a soluble state, and the biological activity of the component protein is reserved to the maximum extent.According to the method, 1,3-propane diamine is produced through the membraneless organelle in escherichia coli for the first time, and the nanoparticles are synthesized in the membraneless organellefor the first time, so that the method is milder and more environment-friendly than a chemical synthesis method.

Description

technical field [0001] The invention relates to a technology in the field of bioengineering, in particular to the construction and application of a membraneless organelle in a prokaryote. Background technique [0002] Compartmentalization is an important physiological process inside cells that confines different metabolic pathways to specific regions within cells. The most common compartments in eukaryotic cells are traditional organelles such as mitochondria and chloroplasts. These organelles have a single or double layer of organelle membranes with a phospholipid bilayer as the skeleton; the most common compartments in prokaryotic cells are called bacterial microbes. Compartment (bacterial microcompartments), which includes a shell composed of proteins and a core region with enzymatic activity. In recent years, researchers have discovered that there are various membraneless organelles in eukaryotic cells. These membraneless organelles are mainly composed of proteins and n...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/62C12N1/21C12P13/00C12R1/19
CPCC12N15/70C12N15/62C07K14/43518C12P13/001C07K2319/00
Inventor 夏小霞韦绍鹏陈梦婷潘芳钱志刚
Owner SHANGHAI JIAO TONG UNIV
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