Method for preparing quantum dot using metal binding protein and recombinant microorganisms therefor

A nanoparticle, protein-binding technology, applied in metallothionein, biochemical equipment and methods, nanotechnology for sensing, etc., can solve the problem of low penetration of optical imaging

Inactive Publication Date: 2009-06-10
KOREA ADVANCED INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Furthermore, to overcome the low in vivo penetration of optical imaging, quantum dots with near-infrared wavelengths were used for optical imaging of sentinel lymph nodes in the axilla of rats (Kim et al., Nat. Biotechnol., 22:93 , 2004)

Method used

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  • Method for preparing quantum dot using metal binding protein and recombinant microorganisms therefor
  • Method for preparing quantum dot using metal binding protein and recombinant microorganisms therefor
  • Method for preparing quantum dot using metal binding protein and recombinant microorganisms therefor

Examples

Experimental program
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Embodiment 1

[0051] Embodiment 1: Preparation of heavy metal binding protein expression vector

[0052] (1) Preparation of Phytochelatin Synthetase Expression Vector

[0053] In order to obtain the AtPCS gene (NCBI accession number AF461180) of synthetic phytochelatin synthase, use the Arabidopsis complementary DNA (cDNA) represented by the sequence (Arabidopsis phytochelatin synthase) by base sequence number: 5 ) as a template and using the primers of SEQ ID NO: 1 and SEQ ID NO: 2 for PCR. The PCR reaction was carried out under the following conditions: pre-denaturation at 94°C for 7 min; denaturation at 94°C for 1 min, cycled 30 times; annealing at 55°C for 1 min; extension at 72°C for 1 min; and finally extension at 72°C for 7 min.

[0054] Serial number: 1:5′-GGAATTCATGGCTATGGCGAGTTTAT-3′

[0055] Serial number: 2:5'-CCCAAGCTTATTAATAGGCAGGAGCAGCGAG-3'

[0056]The 1.5-kb DNA fragment obtained from the PCR reaction was separated by agarose gel electrophoresis, and digested with two ...

Embodiment 2

[0063] Example 2: Fluorescence analysis of E.coli expressing recombinant plasmid pTJ1-AtPCS

[0064] Phytochelatin synthase was prepared by culturing E. coli DH5α transformed with recombinant plasmid pTJ1-AtPCSd with a strongly inducible tac promoter. Based on this purpose, the transformed E.coli was inoculated in a 500mL shake flask containing 100mL LB liquid medium containing heavy metal ions (5mM CdCl 2 2.5H 2 O, ZnCl 2 , SeO 2 、TeCL 4 and CsCl 2 ) and cultured at 37°C. The pTJ1-AtPCS recombinant plasmid has a tac promoter, and IPTG is added to induce gene expression. For this purpose, the culture medium was detected with a spectrophotometer at a wavelength of 600 nm, and when its optical density reached 0.6, 1 mM IPTG was added to induce gene expression. After induction of expression for 4 hours, the medium was centrifuged at 6000 rpm for 5 min at 4°C. Then, discard the supernatant, wash the pellet once with 10 mL PBS buffer (pH 7.4), centrifuge again at 6000 rpm...

Embodiment 3

[0066] Embodiment 3: In vivo heavy metal concentration synthesized by E.coli expressing recombinant plasmid pTJ1-AtPCS feature

[0067] In order to confirm whether the phytochelatin synthase expressed by IPTG can produce heavy metal structures in E.coli DH5α transformed with the recombinant plasmid pTJ1-AtPCS with an induced tac promoter, wash the E.coli in Example 2 with distilled water and dried in a freeze dryer under vacuum for one day, and analyzed the heavy metals accumulated in the cells by TEM (Tehnai G2, FEI, the Netherlands). As a result, the size and shape of heavy metal structures were found to be consistent ( Image 6 ). In addition, it can be observed that the heavy metal structure that does not appear in the control group (a) that does not express phytochelatin synthase appears in (b) in E.coli that expresses phytochelatin synthase ( Image 6 b, 6c and 6d).

[0068] The scale bar in the figure can be used to determine the size of the heavy metal particles...

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Abstract

The present invention relates to a method of preparing heavy metal nanoparticles using a heavy metal-binding protein. More specifically, relates to a method for preparing heavy metal structures, comprising the steps of: culturing a microorganism transformed with a gene encoding a heavy metal-binding protein, in a heavy metal ion-containing medium, to produce heavy metal structures in the microorganism; and collecting the produced heavy metal structures, as well as nanoparticles of heavy metal structures prepared according to said method. Unlike prior methods of preparing quantum dots by physically binding metal materials, the quantum dots disclosed herein can be efficiently produced by expressing the heavy metal-binding protein in cells. In addition, the quantum dots are useful because they can solve an optical stability problem that is the shortcoming of organic fluorophores.

Description

technical field [0001] The present invention relates to a method for preparing heavy metal nanoparticles by using heavy metal binding proteins, in particular to a method for preparing heavy metal structures, the method comprising carrying out the transformation of microorganisms with heavy metal binding protein coding genes in medium containing heavy metal ions culturing, thereby producing heavy metal structures in the microorganisms, and collecting the produced heavy metal structures and nanoparticles of the heavy metal structures produced by the method. Background technique [0002] Quantum dots are nanoscale semiconductor particles that emit light when they are excited by energy such as light, and the color of the emitted light depends on the particle size. That is, if the particle size is reduced thereby reducing the particle dimensionality, the electron cloud density and its energy change, whereby the characteristics of the particle change based on its dimensionality. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N9/02
CPCC12N9/104G01N33/531B82Y15/00G01N33/588C12P3/00B82Y5/00C07K14/825Y10T428/2982C12N15/70C12N15/52
Inventor 李相烨朴泰正
Owner KOREA ADVANCED INST OF SCI & TECH
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