Maltose promoter-containing carrier and maltose promoter mutant
A maltose and promoter technology, applied in the direction of introducing foreign genetic material, DNA/RNA fragments, fermentation, etc. using a vector, can solve problems such as high price and limited application
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[0023] DNA preparation and transformation:
[0024] DNA was isolated from E. coli and B. subtilis or from agarose gels using DNA preparation kits from Tiangen or Omega according to the manufacturer's instructions. Standard molecular techniques were used in all examples. E. coli was transformed using plasmid DNA as described by Chung C.T. et al., Proc. Natl. Acad. Sci. USA 86, 1989, 21722175. According to a modified "Paris method" (Harwood C.R. Molecular Biological Methods for Bacillus, 1990, John Wiley & Sons Ltd., England), plasmid DNA or DNA fragments were used to transform Bacillus subtilis.
[0025] Determination of green fluorescent protein fluorescence intensity RFU:
[0026] Centrifuge at 4°C and 4000rpm for 10 minutes to collect the bacterial cells cultured to an OD600 of about 0.6-0.8, wash the bacterial cells twice with pre-cooled PBS solution, and transfer 150 μL to a 96-well black-bottomed microtiter plate (Corning , USA), placed in a microplate SpectraMax M2 mi...
Embodiment 1
[0035] Example 1 Construction of a pDG vector that lacks the promoter sequence and carries the green fluorescent protein GFP.
[0036] Using the integrated plasmid pDL (BGSC, USA) commonly used in Bacillus subtilis as the backbone, the bgaB gene on the pDL plasmid was replaced by the CPEC PCR cloning method (Nature Protocols, 6, 242–251, 2011, doi: 10.1038 / nprot.2010.181) For the GFP gene, the specific method is to design primers pDG-vector.for / pDG-vector.rev on the upstream and downstream of the bgaB gene of the pDL plasmid to amplify the pDL plasmid by reverse PCR, and simultaneously design primers gfp-insert.for / gfp-insert. rev PCR amplifies the gfp gene from the pSG1729 plasmid (BGSC, USA), wherein the 5' ends of the gfp-insert.for / gfp-insert.rev primers have 20 bp homologous sequences on both sides of the insertion site of the pDL plasmid. After the amplified gfp insert fragment and pDL vector fragment were cut and recovered, they were added to the PCR system at a molar r...
Embodiment 2
[0037] Example 2 Isolation and identification of the MalA promoter derived from the maltose operon of Bacillus subtilis.
[0038] (1) Isolation of the MalA promoter from the maltose operon of Bacillus subtilis.
[0039] Chromosomal DNA of Bacillus subtilis 1A751 was isolated and extracted using Tiangen Bacterial Chromosome Extraction Kit (Beijing, China). Using primers Pglv.for / Pglv.rev, a DNA fragment of about 340 bp between yfiA and MalA genes was obtained by PCR amplification from the chromosomal DNA of Bacillus subtilis 1A751. This fragment was cloned at the upstream of the pDG plasmid green fluorescent protein GFP gene through the BanHI / KpnI site, and the pDG plasmid carrying the maltose operon MalA promoter derived from Bacillus subtilis was obtained, named pDG-G1.
[0040] (2) Identification of the structure of MalA promoter of maltose operon.
[0041] According to the MalA promoter sequence SEQ ID NO: 1 obtained by sequencing, the MalA promoter was analyzed using the...
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