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Komor - GFP-itis Bacterial Base Editor Activity Kit
(Kit # 1000000219 )

Depositing Lab:   Alexis Komor

This collection contains all plasmids needed for preparation and execution of the "GFP-itis" bacterial base editing activity.

This kit will be sent as individual bacterial stabs.

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$240 USD + shipping
Available to academics and nonprofits only.
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Original Publication

Curing “GFP-itis” in Bacteria with Base Editors: Development of a Genome Editing Science Program Implemented with High School Biology Students Vasquez CA, Evanoff M, Ranzau BL, Gu S, Deters E, Komor AC. CRISPR J. 2023 Apr 20; doi: 10.1089/crispr.2023.0002. PubMed (Link opens in a new window) Article (Link opens in a new window).

Description

The "GFP-itis" bacterial base editing activity enables researchers with access to standard microbiology equipment to prepare E. coli with "GFP-itis", a genetic mutation that results in inactive EGFP fluorescence using the GFP-itis pSel plasmid. The GFP-itis pBE-t plasmid encodes a base editor tool that can correct the introduced "GFP-itis" mutation, resulting in EGFP turn on. GFP-itis pBE-nt is provided as a negative control. Both base editor plasmids can be introduced to "GFP-itis" cells in low resource settings, priming this activity for use in research practice partnerships between universities and intermediate high school biology classrooms. The published protocol uses S1030 cells for transformation.

A microcentrifuge tube labelled DNA is shown above three other microcentrifuge tubes (labelled from left to right, E. Coli, KCM, and H2O) all separated by plus signs. A zoomed in panel from the DNA tube reads, “1. Instructors prepare GFP-itis cells, materials, and plasmids” and shows the pBE plasmid containing ABE8e and gRNA features. The pBE-t and pBE-nt gRNA sequences are shown 5’ to 3’ with the pBE-t sequence highlighted and an an underlined. Another zoomed in panel from the E. Coli microcentrifuge tube shows the pSel plasmid containing dGFP with the dsDNA sequence shown and the amino acid sequence above it. The same sequence is highlighted (and the a underlined), on the bottom strand of the dsDNA. Under the 4 microcentrifuge tubes it reads “2. Students chemitransform competent E. Coli with pBE-t and PBE-nt.” An arrow from the 4 microcentrifuge tubes to the right leads to a schematic of the two plasmids and another arrow down, labelled “3. Students plate on LB agar and grow overnight at 37 degrees Celsius”, leads to two photos of a Petri dish visualized with white light or Blue light (~470nm) 580 amber filter. Underneath the Petri dish photos reads “Students visualize plates after 24 hours growth.” The Petri dish is divided in half and streaked with pBE-t on the left and pBE-nt on the right. pBE-t cells glow bright under blue light.
  • Constructs used in the “GFP-itis” activity. The inactivated GFP (dGFP) gene is in the pSel construct (bottom left, yellow background). Shown is the sequence of the dGFP gene, zoomed in on the inactivating A111V mutation that is corrected by editing of the target A to G. The pBE plasmids (top left, blue background) contain the ABE8e editor and one of two gRNA sequences. In pBE-t, the gRNA matches the pSel dGFP mutation site (pink arrow) and will lead to correction of the GFP gene and green fluorescence, while pBE-nt has a non-targeting sequence and acts as a negative control. Instructors prepare all plasmids and, prior to student transformation, incorporate pSel into E. coli to create “GFP-itis cells”. Base editing activity (GFP fluorescence) can be visualized 24 hours post-transformation (shown on the right).

    (Image from bioRxiv, reprinted under the terms of the CC-BY-NC (Link opens in a new window).)

Kit Documentation

The full protocol and supporting materials are included in the publication.

S1030 is the E. coli line used in the kit.

How to Cite this Kit

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.

For your Materials and Methods section:

“The GFP-itis Bacterial Base Editor Activity was a gift from Alexis Komor (Addgene kit #1000000219).”

For your Reference section:

Curing “GFP-itis” in Bacteria with Base Editors: Development of a Genome Editing Science Program Implemented with High School Biology Students Vasquez CA, Evanoff M, Ranzau BL, Gu S, Deters E, Komor AC. CRISPR J. 2023 Apr 20; doi: 10.1089/crispr.2023.0002. PubMed (Link opens in a new window) Article (Link opens in a new window).

The GFP-itis Bacterial Base Editor Activity Kit contains three plasmids. Please refer to the individual plasmid pages below for more details on each plasmid in this kit:

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