CJW4617
(Bacterial strain #177118)

• Purpose: E. coli strain carrying IPTG-inducible gfp-muNS gene for monitoring the mobility of cytoplasmic particles.
• Depositing Lab: Christine Jacobs-Wagner
• Publication: Parry et al Cell. 2014 Jan 16;156(1-2):183-94. doi: 10.1016/j.cell.2013.11.028. Epub 2013 Dec 19.

Backbone

• Vector backbone: none

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): This strain is modified from E. coli MG1655
• Growth instructions: This strain can be grown under normal growth conditions for E. coli. GFP-muNS normally form 1-2 foci per cell when induced with >20uM IPTG in LB or M9 glucose growth media.
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: MG1655 PlacZYA::Plac-gfp-muNS-kan
• Alt name: muNS
• Promoter: Plac
• Tag / Fusion Protein:
  • GFP (N terminal on insert)

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: unknown

Resource Information

• A portion of this plasmid was derived from a plasmid made by: A portion of the gfp-muNS gene was obtained from Max Nibert (Addgene #56657)

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

GFP-muNS expression is under the control of Plac promoter integrated into the endogenous lac locus. The native lac operon (lacZYA) is deleted. Kanamycin resistance casette is inserted downstream of the gfp-muNS gene for cloning purposes.

Cloning method: Lambda RED recombination. The gfp-μNS fragment was amplified from plasmid pER12 (pBAD322A-gfp-μNS), which encodes an N-terminal GFP fusion to residues 471-721 of μNS (Broering et al., 2005) and then digested by KpnI/XhoI. The nptI gene was amplified from pKD4 plasmid and digested by XhoI/SmaI. Then, gfp-μNS and nptI gene fragments were triple ligated into the pKS plasmid. The resulting plasmid was used as a PCR template to generate a gfp-μNS-nptI fragment with flanking regions complementary to lacI and cynX, respectively. This fragment was then used to replace the lac operon (lacZYA) by one-step λ-recombination (Datsenko and Wanner, 2000). The resulting gfp-μNS-nptI replacement of lacZYA was transduced back into MG1655 by P1-phage to generate strain CJW4617. See Parry et al., 2014 for more details. This strain doesn't contain any plasmid.