pHT43 vector (V011875)

Price Information

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

Basic Vector Information

Introducing an inducible expression system into the groESL operon allows for co-expression of a pre-aggregation chaperonin (groESL) and a target protein (bgaB). This system utilizes IPTG to induce co-expression of 80% chaperonin and 20% target protein. Combining this system with the chaperonin-encoding genes htpG and pbpE resulted in 10-13% induced chaperonin. The method was further extended to co-express and identify the amyQα-amylase and the A and B subunits of glutamate synthase using Western blotting. To enhance expression, the chaperonin-protein complex was inserted into a high-copy SD plasmid with multiple cloning sites. The amyQ gene region of α-amylase was inserted into the SD plasmid cloning region, yielding pHT01, which facilitated the expression of a soluble chaperonin-protein complex.

pHT43 vector 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

• Wang G, Guo Z, Zhang X, et al. Heterologous expression of pediocin/papA in Bacillus subtilis. Microb Cell Fact. 2022;21(1):104. Published 2022 May 28. doi:10.1186/s12934-022-01829-x

pHT43 vector Sequence

LOCUS       V011875                 8057 bp    DNA     circular SYN 13-JAN-2022
DEFINITION  Exported.
ACCESSION   V011875
VERSION     V011875
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8057)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8057)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(116..763)
                     /gene="cat"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from
                     Staphylococcus aureus. Accession#: P00485"
     primer_bind     1238..1254
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     CDS             complement(1300..2379)
                     /label="lacI"
                     /note="lac repressor"
     protein_bind    2763..2787
                     /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)."
     promoter        6134..6238
                     /label="AmpR promoter"
     CDS             6239..7096
                     /label="AmpR"
                     /note="beta-lactamase"
     rep_origin      7270..7858
                     /direction=RIGHT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"