A kind of demethylaureomycin biosynthetic gene cluster and its application
A technique of biosynthesis of demethylaureomycin, which is applied in the field of bioengineering and can solve the problems of heavy screening workload, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0042] Example 1 Construction and verification of the recombinant vector pCAPLR carrying the upstream and downstream homology arms of the DMCTC synthetic cluster
[0043] Using the genome of Streptomyces aureus ATCC 13900 (purchased from ATCC) as a template, using primers Larms-up and Larmas-down to amplify the upper homology arms of the demethylaureomycin gene cluster with a size of 1468bp, primers Rarms-up and Rarmas-down The amplified size is 1471bp downstream homology arm of the demethylactetracycline gene cluster. The primer sequences of Rarms-up / Rarmas-down and Larms-up / Larmas-down are respectively shown in SEQ ID No.44-47 and Table 2 in the sequence listing. The enzyme FD KpnI linearized vector plasmid pCAP01 was purchased from GIBCO-BRL Company. Its spectrum is as figure 1 As shown, the plasmid pCAPLR was constructed by the method of homologous recombination, and its map is as follows figure 2 shown. BamHI and XbaI enzyme digestion verification, and then sequencin...
Embodiment 2
[0044] Construction of Example 2 Yeast Transformant LV6-48 (pCAPLRDM)
[0045] Streptomyces aureus ATCC 13900 genome was digested with enzymes FD NsiI and FD MfeI, and the vector pCAPLR prepared in Example 1 was linearized with the restriction site PmeI in the middle of the upstream and downstream homology arms. The digested genome and vector are transformed into yeast LV6-48 protoplasts, and the high homologous recombination ability of yeast is used to finally connect the large fragment of interest to the vector. For the preparation and transformation of yeast protoplasts, see Kouprina, N., et al., Exploring Transformation-Associated Recombination Cloning for Selective Isolation of Genomic Regions. Methods in molecular biology, 2004.255:69-89.
Embodiment 3
[0046] Example 3 Verification of yeast zygote LV6-48 (pCAPLRDM)
[0047] Use primers Larm up-4 and Larm down-4 to group yeast zygote colony PCR against the upstream homology arm, and perform gel electrophoresis for groups with bands and perform colony PCR one by one. Then, primers Rarm up-4 and Rarm down-4 were designed for the downstream homology arm, and primers halo-up and halo-down were designed for the desmethylchlortetracycline gene cluster, and the yeast zygote positive for the first pair of primers was further verified. All 3 pairs of primers are positive for the zygote, after the plasmid is extracted, the plasmid is verified again with these three pairs of primers, and the sequencing result completely matches the expected base sequence. Its spectrum is as image 3 shown. The sequences of primers Halo up / Halo down, Larm up-4 / Larm down-4 and Rarm up-4 / Rarm down-4 are shown in SEQ ID No.48-53 and Table 2, respectively.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More