pLH01 vector (Cat. No.: V016725)
Note: pLH01 is a 9,199 bp plasmid vector designed for genetic engineering in lactic acid bacteria like Lactobacillus plantarum. It features a chloramphenicol resistance marker (12.5 µg/mL) for selection and a p15A origin of replication, making it a low-copy-number vector. It is widely used for CRISPR-based genome editing and heterologous gene expression studies in these bacterial hosts.
- Name:
- pLH01
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 9199 bp
- Type:
- Gene knockout
- Replication origin:
- p15A ori
- Host:
- Lactobacillus plantarum
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 37℃
Resources
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
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
- Huang H, Song X, Yang S. Development of a RecE/T-Assisted CRISPR-Cas9 Toolbox for Lactobacillus. Biotechnol J. 2019 Jul;14(7):e1800690. doi: 10.1002/biot.201800690. Epub 2019 May 20. PMID: 30927506.
pLH01 vector (Cat. No.: V016725) Sequence
LOCUS pLH01 9199 bp DNA circular SYN 26-DEC-2025
DEFINITION Exported.
ACCESSION V016725
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9199)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 9199)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9199
/mol_type="other DNA"
/organism="synthetic DNA construct"
protein_bind 303..336
/label=lox66
/note="Right element (RE) mutant of loxP (Araki et al.,
2010). Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
CDS 579..1226
/gene="cat"
/label=Chloramphenicol acetyltransferase
/note="Chloramphenicol acetyltransferase from
Staphylococcus aureus. Accession#: P00485"
protein_bind 1348..1381
/label=lox71
/note="Left element (LE) mutant of loxP (Araki et al.,
2010). Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
CDS complement(6616..6627)
/label=Factor Xa site
/note="Factor Xa recognition and cleavage site"
rep_origin 8325..8870
/direction=RIGHT
/label=p15A ori
/note="Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin."