Biosynthetic gene cluster for the production of a complex polyketide
A compound and compound technology, applied in organic chemistry, fermentation, DNA/RNA fragments, etc., can solve problems without clear association
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[0096] Suitable preparation techniques are well known to those skilled in the art. See, eg, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor PTess (Cold Spring Harbor, NY). Any of the amino acid sequences provided herein can be readily prepared using a variety of techniques. These and other suitable methods of preparation are well known to those skilled in the art and do not limit the invention.
[0097] In a specific embodiment, the amino acid sequences of the invention are prepared by expressing one or more ORFs or genes in a selected host cell. Typically, vectors are designed to introduce nucleic acid sequences encoding one or more ORFs or genes into desired host cells.
[0098] The terms "vector", "cloning vector" and "expression vector" refer to a vehicle for introducing DNA into a host cell, resulting in the expression of the introduced sequence. In one embodiment, the vector comprises a promoter and one or more control elements (eg, enhancer...
Embodiment 1
[0188] Example 1: Cloning and isolation of Meridamycin biosynthetic gene cluster
[0189] method
[0190] A. Preparation of DNA probes
[0191] A DNA fragment was amplified from genomic DNA of Streptomyces sp. LL-BB0005 by PCR using two pairs of degenerate PCR primers. The first pair of primers was designed based on the conserved amino acid motifs in the ACP and KS domains of type I PKSs. The forward primer (ACP sense) has the sequence 5'-GA(GC)CT(GC)GG(GC)(TC)T(GC)GAC TC(CG)CT(AC)-3' (SEQ ID NO: 2) , the reverse primer (KS antisense) has the sequence 5'-(GC)GA(GC)GA(AG)CA(GC)GC(GC)GT GTC(GC)AC-3' (SEQ ID NO: 3). The second pair of primers was designed based on the highly conserved core motif of the adenylation domain of nonribosomal peptide synthetases. The forward primer (A3 motif) has the sequence 5'-AC(GC)TC(GC)GGC(TA)C(GC)ACCGGC CIG CC(GC)AAG-3' (SEQ ID NO: 4), the reverse primer (A8 motif) has the sequence 5'-AGC TC(GC)A(TC)GC CG(GC)(TA)(GA)G CC(GC)CG(GC)A(TC)CTT(GC...
Embodiment 2
[0198] Example 2: Computational Sequence Analysis of Meridamycin Biosynthesis Gene Cluster
[0199] A. method
[0200] DNA sequence analysis was performed with Lasergene (DNASTAR, Madison, WI) and Vector NTI (InforMax, Frederick, MD). Associations between open reading frames were identified in this gene cluster, and their proposed functions are summarized in Table 1.
[0201] B. Results
[0202] A biosynthetic pathway for meridamycin preparation is proposed based on the cloned gene cluster.
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