Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V008445 | pCM110 | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The plasmid pCM110 is an expression vector specifically developed for use in Methylobacterium extorquens AM1. It is constructed based on an IncP plasmid that lacks both the ColE1 ori and Plac. The construction of pCM110 involves inserting the 0.4 kb HindIII - NsiI fragment of pCM27 containing Pmax into pCM60 (C. J. Marx & M. E. Lidstrom, unpublished results) which had been cut with HindIII and NsiI.
This expression vector provides minimal expression in E. coli, which is advantageous for the introduction of toxic genes into M. extorquens AM1. For example, a construct containing xylE cloned into pCM110 (pCM111) was created to compare its XylE expression level to that of another vector (pCM81) in both organisms. The results showed that pCM111 provided a high level of expression in M. extorquens AM1, 1.5 - 2-fold higher than that achieved with pCM81; however, extracts from E. coli JM109 bearing pCM111 had a basal level of XylE activity, nearly at the background. These results indicate that the Pmax is expressed at very low levels in E. coli. Therefore, while other expression vectors like pCM80 are useful for the expression of most cloned genes, pCM110 may be required to express genes that are toxic in E. coli.
- Vector Name:
- pCM110
- Antibiotic Resistance:
- Tetracycline
- Length:
- 5834 bp
- Type:
- Cloning vector
- Replication origin:
- oriV
- Source/Author:
- Marx CJ, Lidstrom ME.
- Growth Strain(s):
- DH5a
pCM110 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
- Marx CJ, Lidstrom ME. Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria. Microbiology (Reading). 2001 Aug;147(Pt 8):2065-2075. doi: 10.1099/00221287-147-8-2065. PMID: 11495985.
pCM110 vector Sequence
LOCUS 40924_11316 5834 bp DNA circular SYN 17-DEC-2018
DEFINITION Cloning vector pCM110, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5834)
AUTHORS Marx CJ, Lidstrom ME.
TITLE Development of improved versatile broad-host-range vectors for use
in methylotrophs and other Gram-negative bacteria
JOURNAL Microbiology 147 (Pt 8), 2065-2075 (2001)
PUBMED 11495985
REFERENCE 2 (bases 1 to 5834)
AUTHORS Marx CJ, Lidstrom ME.
TITLE Direct Submission
JOURNAL Submitted (12-DEC-2000) Microbiology, University of Washington, Box
357242, Seattle, WA 98195, USA
REFERENCE 3 (bases 1 to 5834)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 5834)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Microbiology 147 (Pt 8), 2065-2075 (2001)"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(12-DEC-2000) Microbiology, University of Washington, Box 357242,
Seattle, WA 98195, USA"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5834
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 3..712
/label=oriV
/note="incP origin of replication"
RBS 820..828
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
regulatory 856..1195
/note="PmxaF; Methylobacterium extorquens mxa operon
promoter"
/regulatory_class="promoter"
CDS complement(1576..2223)
/label=TetR
/note="tetracycline resistance regulatory protein"
CDS 2329..3525
/label=TcR
/note="tetracycline efflux protein"
CDS complement(3762..4907)
/label=trfA
/note="trans-acting replication protein that binds to and
activates oriV"
CDS complement(5179..5550)
/codon_start=1
/gene="traJ"
/product="oriT-recognizing protein"
/label=traJ
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
KQDELGKVMMGVVRPRAEP"
CDS complement(5434..5550)
/codon_start=1
/gene="traJ'"
/product="TraJ'"
/label=traJ'
/note="truncated oriT-recognizing protein"
/protein_id="AAK73417.1"
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL"
gene complement(5434..5550)
/gene="traJ'"
/label=traJ'
oriT complement(5583..5692)
/direction=LEFT
/label=oriT
/note="incP origin of transfer"