Klebsiella for enhanced expression of tqsA gene and application of klebsiella in production of 1,3-propanediol
A Klebsiella, propylene glycol technology, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., can solve the problem that there is no research report on Klebsiella tqsA gene
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Embodiment 1
[0053] The present embodiment provides a construction method for enhancing the expression of tqsA genetically engineered Klebsiella pneumoniae, which comprises the following steps:
[0054] Step 1, using the DNA of the genome sequence of Klebsiella ATCC BAA-830 (the preservation number of the NCBI database is NZ_CP010523.2) as a template, design primers, and the primers are designed as follows:
[0055] Kv-tqsA-F: GGCCGAATTCATGGCTAAACCCATTATTAC (as shown in SEQ ID NO: 1)
[0056] Kv-tqsA-R: GAGCGGATCCTTATTCTTTGCTGACATCAC (as shown in SEQ ID NO: 2),
[0057] The tqsA gene was completely cloned by PCR to obtain the tqsA gene.
[0058] Step 2: Use EcoRI and BamHI to digest the tqsA gene and the expression vector pDK6, connect the tqsA gene to the EcoRI and BamHI restriction sites of the expression vector pDK6, and construct the tqsA enhanced expression plasmid pDK6-tqsA.
[0059] Step 3: Prepare electroporation-competent Klebsiella pneumoniae ATCC 25955, transform the tqsA enha...
Embodiment 2
[0067] The present embodiment provides a construction method for enhancing the expression of tqsA genetically engineered Klebsiella pneumoniae, which comprises the following steps:
[0068] Step 1, using the DNA of the genome sequence of Klebsiella pneumoniae ATCC 25955 (the preservation number of the NCBI database is AQQH00000000) as a template, design primers, and the primers are designed as follows:
[0069] Kp-tqsA-F: GAGCGGATCCATGGCTAAACCCATTATTAC (as shown in SEQ ID NO: 3)
[0070] Kp-tqsA-R: GGCCAAGCTTCTACTGTTTACTGACATCGC (as shown in SEQ ID NO: 4),
[0071] The tqsA gene was completely cloned by PCR to obtain the tqsA gene. Through detection, the nucleotide sequence of the tqsA gene of Klebsiella pneumoniae ATCC25955 and the tqsA gene of Klebsiella mutans ATCC BAA-830 have 90% identity, and the amino acid sequence has 98% identity.
[0072] Step 2: Use BamHI and HindIII to digest the tqsA gene and the expression vector pDK7, connect the tqsA gene to the BamHI and Hin...
Embodiment 3
[0081] The present embodiment provides a construction method for enhancing the expression of tqsA genetically engineered Klebsiella oxytoca, which comprises the following steps:
[0082] Step 1, using the DNA of the genome sequence of Klebsiella pneumoniae ATCC 25955 (the preservation number of the NCBI database is AQQH00000000) as a template, design primers, and the primers are designed as follows:
[0083] Kp-tqsA-F: GAGCGGATCCATGGCTAAACCCATTATTAC (as shown in SEQ ID NO: 3)
[0084] Kp-tqsA-R: GGCCAAGCTTCTACTGTTTACTGACATCGC (as shown in SEQ ID NO: 4),
[0085] The tqsA gene was completely cloned by PCR to obtain the tqsA gene. Through detection, the nucleotide sequence of the tqsA gene of Klebsiella pneumoniae ATCC25955 and the tqsA gene of Klebsiella mutans ATCC BAA-830 have 90% identity, and the amino acid sequence has 98% identity.
[0086] Step 2: Use BamHI and HindIII to digest the tqsA gene and the expression vector pDK7, connect the tqsA gene to the BamHI and HindII...
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