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pLEMO vector (V012824)

Price Information

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V012824 pLEMO 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 Lemo21(DE3) strain contains pLemo, a pACYC184 derivative carrying the lysY gene. Accordingly, chloramphenicol (30 μg/ml) is required to maintain pLemo. In most cases, the T7 promoter-based expression vector will be compatible with pLemo.

Vector Name:
pLEMO
Antibiotic Resistance:
Chloramphenicol
Length:
5003 bp
Type:
E.coli expression plasmid
Replication origin:
p15A ori
Copy Number:
Medium copy number
Promoter:
rhaB
Growth Strain(s):
DH5alpha
Growth Temperature:
37℃
Expression Method:
L-rhamnose induced

pLEMO vector Map

Copy Sequence View Map
pLEMO5003 bp6001200180024003000360042004800rhaRrhaSrhaB promoterT7 lysozymeT7 terminatorCmRcat promoterp15A oriCAP binding site

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

  • Diankristanti PA, Effendi SSW, Hsiang CC, Ng IS. High-level itaconic acid (IA) production using engineered Escherichia coli Lemo21(DE3) toward sustainable biorefinery. Enzyme Microb Technol. 2023 Jun;167:110231.

pLEMO vector Sequence

LOCUS       40924_27663        5003 bp DNA     circular SYN 28-OCT-2023
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5003)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(104..949)
                     /label=rhaR
                     /note="transcriptional activator of rhaR and rhaS"
     CDS             complement(1026..1859)
                     /label=rhaS
                     /note="positive regulator of the rhaB promoter"
     promoter        2005..2123
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             2147..2599
                     /label=T7 lysozyme
                     /note="lysozyme from bacteriophage T7"
     terminator      2672..2719
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2943..3599)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(3600..3702)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(4228..4773)
                     /direction=LEFT
                     /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."
     protein_bind    complement(4964..4985)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."