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pHT43 vector (V011875)

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V011875 pHT43 In stock, instant shipping

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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.

Vector Name:
pHT43
Antibiotic Resistance:
Ampicillin, Chloramphenicol
Length:
8057 bp
Type:
Bacillus subtilis expression vectors
Replication origin:
ori
Copy Number:
High copy number
Promoter:
Pgrac
Cloning Method:
Enzyme digestion and ligation
5' Primer:
LacI-R：GGCATACTCTGCGACATCGT
Fusion Tag:
N-amyQ
Growth Strain(s):
stbl3
Growth Temperature:
37℃

pHT43 vector Map

Copy Sequence View Map
pHT438057 bp400800120016002000240028003200360040004400480052005600600064006800720076008000Chloramphenicol acetyltransferaseM13 fwdlacIPgracSP amyQtrp terminatorParA family proteinhelix-turn-helix domain-containing proteinORF-2AmpR promoterAmpRori

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       Exported                8057 bp DNA     circular SYN 27-AUG-2024
DEFINITION  Exported.
ACCESSION   V011875
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8057)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8057)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8057)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; 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"
     promoter        2683..2802
                     /label=Pgrac
     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)."
     CDS             2809..2901
                     /codon_start=1
                     /label=SP amyQ
                     /translation="MIQKRKRTVSFRLVLMCTLLFVSLPITKTSA"
     terminator      2934..2959
                     /label=trp terminator
     CDS             3023..3286
                     /codon_start=1
                     /label=ParA family protein
                     /translation="MVETYEANIGLLGIVPVLIKSGGSVDQYILDEARKEFGDDLLNST
                     IKLRERIKRFDVQGITEEDTHDKEALKLFNNLTMELIERVEG"
     CDS             3984..5018
                     /codon_start=1
                     /label=helix-turn-helix domain-containing protein
                     /translation="MSEVNLKGNTDELVYYRQQTTGNKIARKRIKKGKEEVYYVAETEE
                     KIWTEEQIKNFSLDKFGTHIPYIEGHYTILNNYFFDFWGYFLGAEGIALYAHLTRYAYG
                     SKDFCFPSLQTIAKKMDKTPVTVRGYLKLLERYGFIWKVNVRNKTKDNTEESPIFKIRR
                     KVPLLSEELLNGNPNIEIPDDEEAHVKKALKKEKEGLPKVLKKEHDEFVKKMMDESETI
                     NIPEALQYDTMYEDILSKGEIRKEIKKQIPNPTTSFESISMTTEEEKVDSTLKSEMQNR
                     VSKPSFDTWFKNTKIKIENKNCLLLVPSEFAFEWIKKRYLETIKTVLEEAGYVFEKIEL
                     RKVQ"
     CDS             complement(5394..5900)
                     /codon_start=1
                     /label=ORF-2
                     /translation="MPYSPTLLEGAVSDILLHKKILSPKDLRPEFLSKRFGIEIMTGKT
                     SYAYIDSDVKVFVLDKKVEEKERNYQFHKLFAHTLLHEENHLEIPMEVYEKHSEEAERL
                     TWVEAIPYHMLRYIDFSDPEFIKQASDVFYIPEKVVQNRINFMIQQQFEIDEFDNVNEK
                     VKSFA"
     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"