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Escherichia coli engineering bacteria and preparation method and applications thereof

A technology of Escherichia coli and engineering bacteria, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of unclear, no literature reports, etc.

Active Publication Date: 2013-09-04
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PcPCS1 Whether the gene has the function of binding heavy metals is not clear, and there is no literature report

Method used

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  • Escherichia coli engineering bacteria and preparation method and applications thereof
  • Escherichia coli engineering bacteria and preparation method and applications thereof
  • Escherichia coli engineering bacteria and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 bean pear PcPCS1 Construction of gene expression vector in Escherichia coli

[0027] bean pear PcPCS1 The construction process of gene Escherichia coli expression vector PcPCS1-pET-22b (+) is as follows figure 1 .

[0028] Pear phytochelatin synthase gene with registration number KC936270 in GenBank PcPCS1 , its coding region nucleotide sequence is SEQ ID NO.1:

[0029] ATGGCGATGG CGGGGTTGTA TCGGCGCCTT CTTCCTTCAC CCCCTGCCGT CGATTTCGCT 60

[0030] TCCTCTCAGG GCAAGCAACT TTTTCTTGAA GCGGTTCAAA ATGGAACCAT GGAAAGCTTT 120

[0031] TACAGGTTGG TTTCATATTT CCAAACGCAA TCAGAGCCTG CATTTTGTGG CCTCGCGAGC 180

[0032] TTGTCCATGG TCCTCAATGC TCTTGCCATT GATCCTGGCA GAAAATGGAA AGGGCCTTGG 240

[0033] AGATGGTTTG ATGAATCTAT GTTAGACTGT TGCGAGCCTT TGGAGACTGT CAAAGTAAGA 300

[0034] GGCATCTCAT TTGGGAAGCT TGTCTGCTTG GCTCACTGTG CTGGAGCAAA AGTTGAAGCC 360

[0035] TTTCGCACAA ATCATAGCAC AATTGATGAG TTTCGTAAAT ATGTATTGAG ATGTTCTACT 420

[0036] TCTGATGATT GTCATGTGAT CTCATCATA...

Embodiment 2

[0071] Embodiment 2 bean pear PcPCS1 Obtaining and Identification of Recombinant Escherichia coli Cells

[0072] Escherichia coli Rosetta (DE3) ( Escherichia coli ) (purchased from Merck, Germany, the company website is http: / / www.emdmillipore.com), graded and streaked on the LB plate medium, and put the streaked plate medium upside down in a constant temperature incubator at 37 °C overnight nourish. Pick a single colony, inoculate it in LB liquid medium, shake it at 150 rpm at 37 °C until OD 600 =0.6, put the cell culture solution in ice to stop the cultivation. Take 1 mL of the above bacteria culture solution in a 1.5 mL centrifuge tube, place on ice for 10 minutes, centrifuge at 10 000 rpm at 4°C for 10 minutes, and discard the supernatant. Add 200 μL of ice-cold 50 mmol / L CaCl 2 The solution was used to gently suspend the cells, placed on ice for 15 minutes, and then centrifuged at 10 000 rpm at 4°C for 10 minutes. Discard the supernatant, add 100 μL of pre-coo...

Embodiment 3

[0075] Example 3 Recombinant Escherichia coli PcPCS1-pET-22b (+) / Rosetta (DE3) to Cd 2+ 、Cu 2+ and Na + determination of tolerance

[0076] The recombinant Escherichia coli strain PcPCS1-pET-22b(+) / Rosetta (DE3) was cultured on LB liquid medium containing 100 mg / L ampicillin to OD 600 = 0.5, while Escherichia coli strain Rosetta (DE3) was used as a control, cultured on LB liquid medium to OD 600 = 0.5. After 10-fold serial dilution of the two bacterial solutions, 5 μL samples were taken and added with different concentrations of CdCl 2 2.5H 2 O, CuCl 2 2H 2 Incubate on LB solid medium with O or NaCl for 16 h at 37°C, observe the growth of the colonies and take pictures.

[0077] Figure 7 A shows: recombinant Escherichia coli strain PcPCS1-pET-22b (+) / Rosetta (DE3) and non-recombinant Rosetta (DE3) bacterial liquid 600 All 0.5) after dilution 1, 10, 100 and 1000 times, the growth on blank LB solid medium was the same.

[0078] Figure 7 B shows: the...

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Abstract

The invention relates to an escherichia coli expression vector PcPcS1-pET-22b (+) of callery pear phytochelatin synthase genes PcPCS1 and an escherichia coli strain PcPCS1-Pet-22b (+) / Rosetta (DE3), and the preservation No. is CGMCCNo. 7569. The phytochelatin synthase genes PcPCS1 are derived from callery pear. On the basis, the invention relates to a method for quantitatively determining callery pear phytochelatin synthase genes absorbing cadmium, copper and sodium based on escherichia coli heterologous-expression and an atomic absorption spectrometry. The endurance capacity and the accumulation capacity of recombined escherichia coli strain PcPCS1-Pet-22b (+) / Rosetta (DE3) to Cd2+, Cu2+ and Na+ are increased, and the recombined escherichia coli strain PcPCS1-Pet-22b (+) / Rosetta (DE3) can be used for microbial remediation for liquid-phase carriers polluted by Cd2+, Cu2+ or Na+ metal ions.

Description

technical field [0001] The present invention relates to the synthesis of soybean pear phytochelatin synthase gene PcPCS1 Constructed on the Escherichia coli expression vector pET-22b(+), and the recombinant plasmid was transformed into the Escherichia coli bacterial strain Rosetta (DE3) ( Escherichia coli ) to obtain recombinant Escherichia coli engineering bacteria PcPCS1-pET-22b(+) / Rosetta (DE3) ( Escherichia coli ), PcPCS1-pET-22b(+) expressed in Rosetta (DE3) made the Escherichia coli strain resistant to Cd 2+ 、Cu 2+ and Na + The tolerance and accumulation ability of Cd can be improved, and the above-mentioned recombinant Escherichia coli engineering bacteria can be used to Cd 2+ or Cu 2+ or Na + The invention relates to the bioremediation of liquid phase carriers polluted by metal ions, and belongs to the field of biotechnology. Background technique [0002] Heavy metal pollution from various industrial and agricultural activities has serious adverse effects on...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/54C02F3/34C12R1/19
Inventor 李慧常有宏蔺经杨青松王中华李晓刚
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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