pNZ8148 vector (V012804)

Price Information

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

Basic Vector Information

pNZ8148 contains an origin of replication (ORI), the gene for the resistance to chloramphenicol, two genes for the replication proteins repA and repC, the nisin-inducible promoter (P nisA), and the transcription terminator (T). The gene, tagged with a Strep-tag II (STREP) followed by a stop codon (*), can be inserted between the necessary NcoI site and another endonuclease site from the multicloning site (MCS) such as PstI, SphI, KpnI, SpeI, XbaI, SacI and HindIII. The replicons of the vectors pNZ8008, pNZ8148, pNZ8149 and pNZ8150 are identical and came originally from the Lactococcus lactis plasmid pSH71. However, this replicon has a broad host range. Plasmids with this replicon can replicate in many Gram-positive bacteria, such as Lactobacillus plantarum and Streptococcus thermophilus. pNZ8148 – In this vector the nisA promoter is followed by an NcoI site for translational fusions at the ATG. It contains a terminator after the MCS. Sequence adaptation for cloning in NcoI can result in a change in the second amino acid of a protein (Mierau and Kleerebezem, 2005). For propagation of pNZ8148, we recommend E. coli strain MC1061 (10 µg/ml chloramphenicol; growth of colonies may take 2 days).

pNZ8148 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.

Note: Use MC1061 competent cells; 10 μg/ml chloramphenicol as antibiotics; Grow the colonies at 30°C and it may take 2 days.

References

• Kazemi-Roudsari, Mahsa et al. “Design of an oral vaccine using Lactococcus lactis against brucellosis: an in vitro and in vivo study.” AMB Express vol. 14,1 2. 3 Jan. 2024, doi:10.1186/s13568-023-01638-4
• Fu, Rui-Yan et al. Wei sheng wu xue bao = Acta microbiologica Sinica vol. 46,3 (2006): 379-84.

pNZ8148 vector Sequence

LOCUS       Exported                3167 bp ds-DNA     circular SYN 17-JUN-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pNZ8148
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3167)
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..3167
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        5..188
                     /note="Pnis"
                     /note="Nisin A promoter region"
     terminator      393..445
                     /note="T"
     CDS             788..997
                     /codon_start=1
                     /note="repC"
                     /note="Replication gene C"
                     /translation="MGGKEANFASVLRPPIKCRVPIFVPKTLYPNWLKGLRGFSIANES
                     PTFSPTFFINLYLSSFIFVFMITK"
     CDS             1266..1964
                     /codon_start=1
                     /note="repA"
                     /note="Replication gene A"
                     /translation="MAIKNTKARNFGFLLYPDSIPNDWKEKLESLGVSMAVSPLHDMDE
                     KKDKDTWNSSDVIRNGKHYKKPHYHVIYIARNPVTIESVRNKIKRKLGNSSVAHVEILD
                     YIKGSYEYLTHESKDAIAKNKHIYDKKDILNINDFDIDRYITLDESQKRELKNLLLDIV
                     DDYNLVNTKDLMAFIRLRGAEFGILNTNDVKDIVSTNSSAFRLWFEGNYQCGYRASYAK
                     VLDAETGEIK"
     CDS             2424..3074
                     /codon_start=1
                     /note="cm"
                     /note="chloramphenicol resistant gene"
                     /translation="MNFNKIDLDNWKRKEIFNHYLNQQTTFSITTEIDISVLYRNIKQE
                     GYKFYPAFIFLVTRVINSNTAFRTGYNSDGELGYWDKLEPLYTIFDGVSKTFSGIWTPV
                     KNDFKEFYDLYLSDVEKYNGSGKLFPKTPIPENAFSLSIIPWTSFTGFNLNINNNSNYL
                     LPIITAGKFINKGNSIYLPLSLQVHHSVCDGYHAGLFMNSIQELSDRPNDWLL"