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About Gateway® Custom Knockout Clones

General Information

The PFGRC is making available, at no cost to researchers, Gateway® Knock-out Clones. In order for the PFGRC to efficiently utilize its resources, the following guidelines have been developed:

  • The PFGRC will accept orders for up to 12 Gateway® Knock-out Clones from any individual investigator.
  • Clones are comprised of a left flank, selectable marker, and a right flank (see Gateway® MultiSite Pro Strategy below).
  • The selectable marker's entry clone (replacement gene) will be sequence confirmed and the final three-fragment construct will be confirmed for presence and orientation of all components by restriction digestion.
  • Annotations are based on current RefSeq entries for sequenced strains
  • We provide clones with a Gateway destination plasmid containing the three-fragment construct. We do not provide the deletion mutant strain.

Available Organisms/strains

  • Aspergillus fumigatus Af1163
  • Mycobacterium tuberculosis CDC1551

Aspergillus fumigatus Af1163 Premade Knockouts Pilot

The PFGRC is making available seventy Aspergillus fumigatus Af1163 knock-out clones for targeted gene replacement. These clones were the result of a pilot project with the Aspergillus research community to rapidly create 96 knock-out constructs by adapting Invitrogen’s Gateway® MultiSite technology to our high-throughput entry cloning platform.

Using the three-fragment MultiSite strategy (Cat# 12537-103) the pyrG selectable marker was assembled with chromosomal amplicons (~1Kb) of the regions flanking the targeted gene. The three fragments (left flank, pyrG, right flank) were amplified and cloned into entry vectors pDONR™ 221 P1-P4, pDONR™ 221 P4r-P3r, and pDONR™ 221 P3-P2 respectively. The flanking clones were confirmed by PCR, the pyrG clone was sequence verified. The final assembled construct is created using LR Clonase™ II Plus to recombine the three clones with each other and the destination vector, pET-DEST-TIGR02.

Linearized vector can be used to transform chemically competent protoplasts. Upon homologous recombination the pyrG gene complements an auxotrophic mutant Af1163 strain.

Mycobacterium tuberculosis CDC1551 Knockout Project

Using the three-fragment MultiSite Pro strategy (Invitrogen, Cat# 12537-103) a PCR product representing a functional hygromycin resistance cassette was assembled with chromosomal amplicons of approximately 600 bp of the regions flanking each gene targeted for replacement. The three fragments (left flank, hygromycinR, right flank) were amplified and cloned into the entry vectors pDONR™ 221 P1-P4, pDONR™ 221 P4r-P3r, and pDONR™ 221 P3-P2, respectively. The flanking clones were confirmed by restriction digestion analysis.

The hygromycin resistance cassette was sequence verified in addition to experimentally verified to confer hygromycin resistance to DH10B-T1 E. coli cells. The final, assembled MultiSite construct was created using LR Clonase™ II Plus for the ordered assembly and cloning of each insert into the cosmid cloning vector pDEST-YUB. Each final destination construct was verified by restriction digest analysis.

The pDEST-YUB destination vector was supplied by Scott Nolan at Johns Hopkins University. This vector represents a Gateway® compatible adaptation of the cloning vector pYUB584, a previously described cosmid cloning vector (Bardarov, 2002). Each destination construct that we have created, then, can be used in the Mycobacterium specialized transduction method that Bardarov and colleagues have described.

Selectable Marker (provided by investigator)

As we launch this service we are asking investigators to provide the PFGRC with the desired selectable marker (DNA material and sequence) for targeted gene replacement via homologous recombination. In addition to investigator contributions, we are actively seeking suitable selectable markers from published sources to offer a la carte marker selection in the near future.

All Aspergillus fumigatus Af1163 knockouts will be created with pyrG as the selectable marker.

All markers submitted:

  • Should be thoroughly tested and sequenced
  • Contain suitable characteristics (constitutive promoter, terminatorless, etc.) for expression in mutant strains minimizing polar effects.
  • Be compatible with organism/strain
  • Becomes a resource of the PFGRC (MTA required)

Ordering

  1. Click Order Custom Clones in left menu.
  2. Select Organism/strain from offering list (note Knock-out availability)
  3. Choose Knock-out clones option
  4. Select desired clones from order form or further filter list
  5. Click Add to Cart
  6. Checkout and supply contact/shipping information
  7. Sign and mail in appropriate documentation (see below)

Expectations for the Investigator

The following documentation must be submitted and approved before you can receive clones:

The PFGRC strongly encourages the timely deposition of derived Knock-out strains into a suitable community repository (e.g. Fungal Genetics Stock Center)

Delivery Timeframe:

~8 weeks from receipt of selectable marker DNA and sequence (also dependent on receiving MTA/CofC approval)

Package Contents*

  • E. coli glycerol stocks containing Gateway® knock-out clones.
  • Data CD with:
    • Order Summary and plate/tube map
    • Validation report (HTML)
    • Knockout clone support document
    • Licensing agreement

* Clones are shipped on dry ice. The receiving facility must be equipped to transfer the clones to cold storage immediately upon receipt.

Gateway® MultiSite Pro Strategy