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Pan-emergency protein fauspa gene, expression vector and its construction and application derived from extreme acidophilus

A pan-emergency protein, shuttle expression vector technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve problems affecting the absorption of sugar and amino acids

Inactive Publication Date: 2016-02-10
SHANGHAI ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that the toxicity of butanol is mainly to destroy the phospholipid components of the cell membrane, which further affects the absorption of sugar and amino acids

Method used

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  • Pan-emergency protein fauspa gene, expression vector and its construction and application derived from extreme acidophilus
  • Pan-emergency protein fauspa gene, expression vector and its construction and application derived from extreme acidophilus
  • Pan-emergency protein fauspa gene, expression vector and its construction and application derived from extreme acidophilus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 Synthesis of the pan-emergency protein FaUSPA gene derived from the extreme acidophilus Ferroplasmaacidarmanus

[0017] On the premise of not changing the amino acid sequence, according to the specific codon characteristics of Clostridium, the pan-emergency protein FaUSPA gene (GenbankZP_05570298.1) from the extreme acidophilic bacteria Ferroplasmaacidarmanus was synthesized, and the primers were designed and artificially synthesized. The primer sequence is as follows, and the primer length is about 60bp.

[0018] 1. N1100=FAUSPAI-1:Tm=54,60mer

[0019] GGA,TCC,ATG,AAG,GTC,CTC,GTC,TCC,TAT,GAT,GGT,TCT,GAA,GGT,GCT,AGA,GAA,GCT,CTC,AAG

[0020] 2. N1100=FAUSPAI-2: Tm=54,60mer

[0021] TGT, AGT, ACT, CGT, CAA, CAG, TGC, ACT, TGA, GTT, CAA, GAG, CAT, ACT, TGA, GAG, CTT, CTC, TAG, CAC

[0022] 3. N1100=FAUSPAI-3:Tm=54,60mer

[0023] CAC,TGT,TGA,CGA,GTA,CTA,CAT,CGT,CTA,CGT,CAT,TCC,AAC,TAT,CGT,TGG,TAT,CTC,TGC,TTC

[0024] 4. N1100=FAUSPAI-4:Tm=54,60mer

[0025] C...

Embodiment 2

[0042] Example 2 Construction of Escherichia coli-clostridium shuttle expression vector containing FaUSPAI gene

[0043] Based on the pSOS94 vector (Desai1999, ApplEnvironMicrobiol), the chloramphenicol resistance gene expression unit, the Clostridium CAC3645 gene (NC_003030) promoter, the FaUSPAI gene reading frame expression unit and the Bacillus subtilis pyruvatekinase (PYK, NC_000964) gene termination Substitute the ctfAB and abc expression units in the pSOS94 vector after the directional connection of the promoter (PYK), and construct the Clostridium-E. figure 1 ), the vector is about 6670bp. The specific construction steps are as follows:

[0044] The chloramphenicol expression unit was amplified from pXYP251 (GenBank accession number AY178046, containing a medium-strength prokaryotic PGm promoter and terminator T1T2) vector. The primers are cm1:5'-AAGCTTATAACTTCGTATAATGTATGCTATACGAAG-3'; cm2:5'-TCTAGATATACGAAGATAACTTCGTATAGCATACAT-3'. The amplification conditions wer...

Embodiment 3

[0049] Embodiment 3 transformation plasmid treatment and transformation method

[0050] The Escherichia coli-clostridium shuttle expression vector pFaUSPA constructed above contains resistance to ampicillin and erythromycin, can replicate in Escherichia coli, and can screen transformed Clostridium by erythromycin. The shuttle expression vector is methylated using the methylase on the pAN1 plasmid (Mermelstein1993, ApplEnvironMicrobiol), which contains the δ3T methyltransferase gene from Bacillus subtilis, which can realize the methylation protection of the Clostridium expression vector, thereby Avoid it being cleaved by the Cac824I restriction endonuclease in Clostridium, because this restriction site is ubiquitous in Clostridium.

[0051] The E. coli used for the amplification of the expression vector pFaUSPA must use a methylase-deficient strain, because when some cytosine methylated DNA is transformed into common E.coli strains, the transformation efficiency will be signifi...

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Abstract

The invention relates to a universal stress protein FaUSPA gene derived from ferroplasma acidarmanus, an expression vector as well as construction and an application thereof. A nucleotide sequence of the universal stress protein FaUSPA gene is shown as SEQ ID NO. 1. Through constructing an escherichia coli-clostridium shuttling expression vector including the universal stress protein FaUSPA gene, tests indicate that the universal stress protein FaUSPA gene can be stably and efficiently expressed in clostridium, and thus the tolerance of the clostridium to the butanol is effectively improved. Meanwhile, by using anaerobic fermentation, the gene is capable of greatly improving the capacity of producing the butanol by the clostridium.

Description

technical field [0001] The invention relates to a pan-emergency protein FaUSPA gene derived from the extreme acidophilic bacterium Ferroplasmaacidarmanus, an Escherichia coli-clostridium shuttle expression vector containing the gene and a construction method thereof, and their role in improving the butanol tolerance of Clostridium and promoting the shuttle The application of bacteria to improve the production of high-content butanol. Background technique [0002] Bio-butanol is a second-generation new biofuel with great potential. Compared with ethanol, butanol has the following advantages as a biofuel: 1) High energy content, which can travel 30% more than ethanol ; 2) The volatility of butanol is only 1 / 6 times that of ethanol and 1 / 13.5 of that of gasoline. It has a large tolerance to water when mixed with gasoline, and has better adaptability to humidity and low water vapor pressure; 3) Butanol Can be used in existing fuel supply and distribution systems, while ethanol ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/74C12N1/21C12R1/145
Inventor 彭日荷姚泉洪田永生赵伟付晓燕朱波许晶高建杰韩红娟王波王丽娟韩静薛永
Owner SHANGHAI ACAD OF AGRI SCI