Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V004412 | pMTL85151 | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pMTL85151 is a shuttle plasmid designed for constructing allelic exchange vectors. It facilitates genetic manipulation within selected Clostridium perfringens strains exhibiting high transformation efficiency, thereby validating its utility in enhancing gene transfer efficacy.
- Vector Name:
- pMTL85151
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 3729 bp
- Type:
- Shuttle vector
- Replication origin:
- ori
- Source/Author:
- Heap JT, Pennington OJ, Cartman ST, Minton NP.
- Growth Strain(s):
- JM109
pMTL85151 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
- Grosse-Honebrink A, Schwarz KM, Wang H, Minton NP, Zhang Y. Improving gene transfer in Clostridium pasteurianum through the isolation of rare hypertransformable variants. Anaerobe. 2017 Dec;48:203-205. doi: 10.1016/j.anaerobe.2017.09.001. Epub 2017 Sep 9. PMID: 28899647; PMCID: PMC5734229.
pMTL85151 vector Sequence
LOCUS V004412 3729 bp DNA circular SYN 18-DEC-2018
DEFINITION Exported.
ACCESSION V004412
VERSION V004412
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 3729)
AUTHORS Heap JT, Pennington OJ, Cartman ST, Minton NP.
TITLE A modular system for Clostridium shuttle plasmids
JOURNAL J. Microbiol. Methods 78 (1), 79-85 (2009)
PUBMED 19445976
REFERENCE 2 (bases 1 to 3729)
AUTHORS Pennington OJ, Heap JT, Cartman ST, Minton NP.
TITLE Direct Submission
JOURNAL Submitted (02-MAR-2009) Centre for Biomolecular Sciences, The
University of Nottingham, University Park, Nottingham NG7 2RD,
United Kingdom
REFERENCE 3 (bases 1 to 3729)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 3729)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J.
Microbiol. Methods"; date: "2009"; volume: "78"; issue: "1"; pages:
"79-85"
SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(02-MAR-2009) Centre for Biomolecular Sciences, The University of
Nottingham, University Park, Nottingham NG7 2RD, United Kingdom"
SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..3729
/mol_type="other DNA"
/organism="synthetic DNA construct"
regulatory 9..62
/note="terminator derived from CD0164 gene of Clostridium
difficile"
/regulatory_class="terminator"
primer_bind 63..79
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(199..215)
/label="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
regulatory 363..404
/note="terminator derived from fdx gene of Clostridium
pasteurianum"
/regulatory_class="terminator"
CDS 783..1223
/codon_start=1
/gene="repL"
/product="plasmid replication protein"
/label="repL"
/protein_id="ACR43917.1"
/translation="MKERYGTVYKGSQRLIDEESGEVIEVDKLYRKQTSGNFVKAYIVQ
LISMLDMIGGKKLKIVNYILDNVHLSNNTMIATTREIAKATGTSLQTVITTLKILEEGN
IIKRKTGVLMLNPELLMRGDDQKQKYLLLEFGNFEQEANEID"
gene 783..1223
/gene="repL"
/label="repL"
CDS 1373..1993
/gene="catP"
/label="Chloramphenicol acetyltransferase"
/note="Chloramphenicol acetyltransferase from Clostridium
perfringens. Accession#: P26826"
rep_origin 2173..2761
/direction=RIGHT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
oriT 3082..3191
/label="oriT"
/note="incP origin of transfer"
CDS 3224..3592
/label="traJ"
/note="oriT-recognizing protein"