pWB980-ori (pUC980-2) vector (Cat. No.: V017497)

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Basic Information

Note: pUC980-2 is a high-copy-number and highly stable E. coli-B. subtilis shuttle expression plasmid. It was constructed by inserting the E. coli replication origin (ori) upstream of the BA3-1 region in the pWB980-DB backbone. In B. subtilis, it maintains over 450 copies per cell with a segregational stability of up to 100%. It has been successfully used for the high-yield secretory production of heterologous proteins like alkaline pectate lyase (PelN) and alkaline protease (Spro1) in B. subtilis.

pWB980-ori (pUC980-2) is a shuttle plasmid with an additional replication origin compared to pWB980.

Name:: pWB980-ori (pUC980-2)

Antibiotic Resistance:: Kanamycin

Length:: 4276 bp

Type:: Bacillus Expression vector

Copy Number:: High copy number

Promoter:: P43

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

$ 199.3

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5. Store the plasmid at -20 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

References

Zhao X, Xu J, Tan M, Zhen J, Shu W, Yang S, Ma Y, Zheng H, Song H. High copy number and highly stable Escherichia coli-Bacillus subtilis shuttle plasmids based on pWB980. Microb Cell Fact. 2020 Feb 7;19(1):25. doi: 10.1186/s12934-020-1296-5. PMID: 32028973; PMCID: PMC7006159.