Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V008303 | pCold TF | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The complexity of the transformation operation was avoided by the development of pCold TF DNA, a fusion cold shock expression vector with the E. coli chaperone protein Trigger Factor (TF) as a soluble tag.TF has a molecular weight of 48 kDa, and Trigger Factor is a prokaryotic ribosome-binding chaperone protein that promotes co-translation and folding of nascent peptide chains.TF is derived from E. coli and has a high efficiency of expression compared to other biologically-derived tags.TF has a high efficiency of expression compared to other biologically-derived tags. Folding.TF is derived from E. coli and is expressed in E. coli with high efficiency and solubility compared to tags from other biological sources. This vector inserts a 5' non-coding region (5'UTR), TEE (Translation Enhancement Element) sequence, His-Tag sequence, TF sequence, and Multiple Cloning Site (MCS) downstream of the cspA promoter. A lac operator was also inserted downstream of the cspA promoter to tightly regulate protein expression. And cut-off sites for HRV 3C Protease, Thrombin and Factor Xa are inserted between the TF sequence and MCS, which are used to eliminate tags of fusion proteins. pCold TF DNA applies the promoter of E. coli, and like the other pCold DNAs, most of E. coli can be used as an expression host. Using pCold TF DNA, soluble expression of difficult-to-express genes can be achieved through the soluble tagging function of TF and the molecular chaperone effect.
- Vector Name:
- pCold TF
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5769 bp
- Type:
- Expression vector
- Replication origin:
- ori
- Source/Author:
- Shodai T, Takakura H.
pCold TF 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
- Sui B, Wang X, Zhao T, Zhen J, Ren H, Liu W, Zhang X, Zhang C. Design, Screening, and Characterization of Engineered Phage Endolysins with Extracellular Antibacterial Activity against Gram-Negative Bacteria. Appl Environ Microbiol. 2023 Jul 26;89(7):e0058123. doi:10.1128/aem.00581-23. Epub 2023 Jun 20. PMID:37338346; PMCID: PMC10370328.
pCold TF vector Sequence
LOCUS 40924_12715 5769 bp DNA circular SYN 17-DEC-2018
DEFINITION Expression vector pColdTF DNA, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5769)
AUTHORS Shodai T, Takakura H.
TITLE Expression vector pColdTF
JOURNAL Published Only in Database (2005)
REFERENCE 2 (bases 1 to 5769)
AUTHORS Shodai T, Takakura H.
TITLE Direct Submission
JOURNAL Submitted (17-MAY-2005) Toshihiro Shodai, TAKARA BIO INC.;
Seta3-4-1, OTSU, SHIGA 520-2193, Japan
(E-mail:shiyoudait@takara-bio.co.jp, Tel:81-77-543-7229,
Fax:81-77-543-7238)
REFERENCE 3 (bases 1 to 5769)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 5769)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Published
Only in Database (2005)"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(17-MAY-2005) Toshihiro Shodai, TAKARA BIO INC."; volume: "
Seta3-4-1, OTSU, SHIGA 520-2193, Japan
(E-mail:shiyoudait@takara-bio.co.jp, Tel:81-77-543-7229, Fax";
pages: "81-77-543-7238"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5769
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 15..81
/label=cspA promoter
/note="promoter of the E. coli cold shock protein cspA gene
(Mitta et al., 1997)"
protein_bind 84..100
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
5'UTR 122..255
/label=cspA 5'UTR
/note="5'UTR of the E. coli cold shock protein cspA gene
(Mitta et al., 1997)"
CDS 256..270
/label=TEE
/note="translation enhancing element for E. coli (Qing et
al., 2004)"
CDS 271..288
/label=6xHis
/note="6xHis affinity tag"
CDS 289..1584
/label=Trigger Factor
/note="ribosome-associated chaperone from E. coli (Hoffmann
et al., 2010)"
CDS 1594..1617
/label=HRV 3C site
/note="recognition and cleavage site for human rhinovirus
3C and PreScission proteases"
CDS 1627..1644
/label=thrombin site
/note="thrombin recognition and cleavage site"
CDS 1651..1662
/label=Factor Xa site
/note="Factor Xa recognition and cleavage site"
misc_feature 1663..1722
/gene="tig"
/note="MCS = Multiple cloning sites contain the follow
restriction sites: NdeI, SacI, KpnI, XhoI, BamHI, EcoRI,
HindIII, SalI, PstI, XbaI"
3'UTR 1730..1874
/label=cspA 3'UTR
/note="3'UTR of the E. coli cold shock protein cspA gene
(Mitta et al., 1997)"
rep_origin complement(2073..2528)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2628..2732
/label=AmpR promoter
CDS 2733..3590
/label=AmpR
/note="beta-lactamase"
rep_origin 3764..4352
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind complement(4490..4511)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(4527..5606)
/label=lacI
/note="lac repressor"
promoter complement(5607..5684)
/label=lacI promoter