Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V011282 | pSIP403 | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The pSIP403 is for expression of gene of interest in Lactobacillus plantarum and L. sakei. sppK perceives an extracellular signal and autophosphorylates. It then transfers the phosphate to sppR. The phosphorylated sppR binds to the sppA promoter, activating it and initiating transcription of the sppA gene. Erythromycin was added as follows: 200 μg/mL for E. coli and 10 μg/mL for lactobacilli.
- Vector Name:
- pSIP403
- Antibiotic Resistance:
- Erythromycin
- Length:
- 7435 bp
- Type:
- Bacterial Expression
- Replication origin:
- ori
- Promoter:
- sppA
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- GGCTTTTATAATATGAGATAATGCCGAC
- Growth Strain(s):
- JM108
- Growth Temperature:
- 37℃
pSIP403 vector Map
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
- Sørvig E, Mathiesen G, Naterstad K, Eijsink VGH, Axelsson L. High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors. Microbiology (Reading). 2005 Jul;151(Pt 7):2439-2449.
pSIP403 vector Sequence
LOCUS Exported 7435 bp DNA circular SYN 21-APR-2025
DEFINITION Exported.
ACCESSION V011282
VERSION .
KEYWORDS pSIP403
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7435)
AUTHORS Sorvig E, Mathiesen G, Naterstad K, Eijsink VG, Axelsson L
TITLE High-level, inducible gene expression in Lactobacillus sakei and
Lactobacillus plantarum using versatile expression vectors.
JOURNAL Microbiology. 2005 Jul;151(Pt 7):2439-49. doi:
10.1099/mic.0.28084-0.
PUBMED 16000734
REFERENCE 2 (bases 1 to 7435)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7435)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 7435)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Microbiology."; date: "2005-07"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7435
/mol_type="other DNA"
/organism="synthetic DNA construct"
source join(542..7435,1..541)
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 1..672
/codon_start=1
/product="Encodes a response regulator. Receives the
phosphate group from phosphorylated sppK, then binds to DNA
to regulate gene transcription."
/label=SppR
/translation="MINEFAMTLTCAASDTETLLAAIKDQQRGLFFLDMEIEDNRQAGL
EVATKIRQMMPFAQIVFITTHEELTLLTLERKIAPLDYILKDQTMAEIKRQLIDDLLLA
EKQNEAAAYHRENLFSYKIGPRFFSLPLKEVVYLYTEKENPGHINLLAVTRKVTFPGNL
NALEAQYPMLFRCDKSYLVNLSNIANYDSKTRSLKFVDGSEAKVSFRKSRELVAKLKQM
M"
promoter 972..1115
/label=PsppA
/note="A DNA sequence upstream of the sppA gene. Inactive
without an inducing signal; activates gene transcription
when stimulated."
CDS 1116..2915
/label=GUS
/note="beta-glucuronidase"
rep_origin 3272..3860
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature 4032..4719
/label=256rep
/note="p256 replication region; allows replication in
Lactobacillus species"
CDS 5049..5783
/gene="ermBP"
/label=rRNA adenine N-6-methyltransferase
/note="rRNA adenine N-6-methyltransferase from Enterococcus
faecalis. Accession#: P0A4D5"
CDS 6013..7359
/codon_start=1
/product="Encodes a histidine-kinase in a two-component
system. It senses extracellular signals (like pheromones,
IP-673) and autophosphorylates upon signal binding."
/label=SppK
/translation="MLYTDVSVSLMQNFVAILLIFLLYRYIQRKITFKRIILDILIAII
FSILYLFISDASLLVMVLMRLGWHFHQQKENKIKTTDTANLILIIVIQLLLVAVGTIIS
QFTISIIKSDFSQNILNNSATDITLLGIFFAVLFDGLFFILLKNKRTELQHLNQEIIEF
SLEKQYFIFIFILFIVIEIILAVGNLQGVTATILLTIIIIFCVLIGMTFWQVMLFLKAY
SIRQEANDQLVRNQQLQDYLVNIEQQYTELRRFKHDYQNILLSLESFAEKGDQQQFKAY
YQELLAQRPIQSEIQGAVIAQLDYLKNDPIRGLVIQKFLAAKQAGVTLKFEMTEPIELA
TANLLTVIRIIGILLDNAIEQAVQETDQLVSCAFLQSDGLIEITIENTASQVKNLQAFS
ELGYSTKGAGRGTGLANVQDLIAKQTNLFLETQIENRKLRQTLMITEET"