pKJE7 vector (V012942)

Price Information

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V012942 pKJE7 In stock, instant shipping

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

The pKJE7 vector plasmid is a chaperone plasmid. Primarily for facilitating the production and folding of recombinant proteins in bacterial systems, particularly Escherichia coli (E. coli). It has been shown that the co-expression of ScFvs and the chaperone plasmids PG-tf2, ptf16, pGro7, pKJE7, and pG-KJE8 leads to a noticeable increase in the expression of soluble proteins.

Vector Name:
pKJE7
Antibiotic Resistance:
Chloramphenicol
Length:
7192 bp
Type:
Packaging assistance
Replication origin:
p15A ori
Host:
E. coli
Growth Strain(s):
DH10B
Growth Temperature:
37℃

pKJE7 vector Map

pKJE77192 bp30060090012001500180021002400270030003300360039004200450048005100540057006000630066006900CmRChaperone protein DnaKChaperone protein DnaJProtein GrpEp15A oricat promoter

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

  • Nazari A, Farajnia S, Zahri S, Bagherlou N, Tanoumand A, Rahbarnia L. Cytoplasmic Chaperones Enhance Soluble Expression of Anti-EGFR huscFv in Escherichia coli. Iran J Biotechnol. 2020 Apr 1;18(2):e2314. doi: 10.30498/IJB.2020.138200.2314. PMID: 33542937; PMCID: PMC7856399.
  • Yasamut U, Thongheang K, Weechan A, Sornsuwan K, Juntit OA, Tayapiwatana C. Evaluating the ability of different chaperones in improving soluble expression of a triple-mutated human interferon gamma in Escherichia coli. J Biosci Bioeng. 2024;138(3):232-238. doi:10.1016/j.jbiosc.2024.06.005
  • Rani RM, Syngkli S, Nongkhlaw J, Das B. Expression and characterisation of human glycerol kinase: the role of solubilising agents and molecular chaperones [published correction appears in Biosci Rep. 2023 Oct 31;43(10):BSR-2022-2258_COR. doi: 10.1042/BSR-2022-2258_COR]. Biosci Rep. 2023;43(4):BSR20222258. doi:10.1042/BSR20222258

pKJE7 vector Sequence

LOCUS       Exported                7192 bp DNA     circular SYN 10-OCT-2024
DEFINITION  Exported.
ACCESSION   V012942
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7192)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7192)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7192
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1787..3700
                     /gene="dnaK"
                     /label=Chaperone protein DnaK
                     /note="Chaperone protein DnaK from Escherichia coli O1:K1 /
                     APEC. Accession#: A1A766"
     CDS             3792..4919
                     /gene="dnaJ"
                     /label=Chaperone protein DnaJ
                     /note="Chaperone protein DnaJ from Escherichia coli (strain
                     K12). Accession#: P08622"
     CDS             4939..5529
                     /gene="grpE"
                     /label=Protein GrpE
                     /note="Protein GrpE from Escherichia coli (strain K12). 
                     Accession#: P09372"
     rep_origin      5800..6345
                     /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."
     promoter        6871..6973
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     CDS             join(6974..7192,1..438)
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"