11 Matching Annotations
  1. Mar 2023
    1. Vmax has a low-level natural resistance to kanamycin but is more susceptible to other antibiotics. When selecting on Kan plates, Vmax with KanR will simply grow faster than Vmax without KanR. Larger colonies=KanR. Other antibiotics select against Vmax but must have a dosage change compared to E. coli.
    2. Wash the culture in fresh LB before adding glycerol, to avoid problems with reviving the culture later

      To prepare V. natriegens cells for −80 °C storage, an overnight culture of V. natriegens was washed in fresh medium before storage in glycerol. Cultures were centrifuged for 1 min at 20,000g and the supernatant was removed. The cell pellet was resuspended in fresh LB3 medium and glycerol was added to 20% final concentration. The stock was quickly vortexed and stored at −80 °C. Bacterial glycerol stocks stored in this manner are viable for at least five years.

      source: Lee, Henry H., et al. "Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi." Nature microbiology 4.7 (2019): 1105-1113.

    1. Four inducible promoters were found to function in V. natriegens

      Lac, Pbad (arabinose), lambda promoter

    2. Vmax@SGIDNA.com

      Is the genotype of Vmax proprietary? I could not locate any references in the SGI website as well

    1. For best results, use only LB-Miller agar plates. Do not use LB-Lennox or LB-Luriaplates (Vmax™ growth will be suboptimal).
    2. Vibrio natriegens strain containing a major extracellular nuclease knockout and insertion of an IPTG-inducible T7 RNA polymerase cassette for expression of genes under a T7 promoter.

      dns nuclease- (Gibson et al)

    1. we successfully demonstrated challenging clonings such as Gibson Assembly with 5 and Aquacloning with 3 fragments as well as 8 part golden-gate reactions.
    1. all downstream manipulations with V. natriegens ATCC14048 Δdns cells were performed with antibiotic selection (kanamycin (200 μg/mL), chloramphenicol (2 μg/mL), carbenicillin (100 μg/mL))