pYS1
(Plasmid #117488)

• Purpose: Generation of unmarked gene deletions in Mycobacterium smegmatis
• Depositing Lab: Eyal Gur
• Publication: Shenkerman et al Gene. 2014 Jan 1;533(1):374-8. doi: 10.1016/j.gene.2013.09.082. Epub 2013 Oct 4.

Backbone

• Vector backbone: pYS1
• Vector type: Deletion generation

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): DH5alpha
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: Che9c 60–61, sacB, aph
• Species: M. smegmatis
• Promoter: Acetamide

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: CGGTACCAGATCTTTAAA
• 3′ sequencing primer: CCAGTGTTACAACCAATTA

Resource Information

• Supplemental Documents:
  • pYS1.gb

Terms and Licenses

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

Depositor Comments

Addgene QC Next-generation sequencing identified sequence discrepancies relative to the Genbank file linked under Supplemental Documents. Plasmid is expected to function as described in the associated publication.

Detailed knockout procedure

• Transform plasmid pYS1 into MC2155 cells and grow at 34 °C in liquid medium containing kanamycin.

• When cells reach exponential phase, add acetamide (0.2%, w/v) to induce Che9c 60–61 expression overnight.

• Prepare competent cells and store at − 80 °C until further use.

• Construct the recombineering gfp-hyg linear DNA fragment by cloning into pYS2 upstream and downstream flanking regions (~ 700 bp each) of the gene to be deleted.

• Using appropriate restriction enzymes, excise your recombineering gfp-hyg linear DNA fragment from pYS2, transform into the Che9c 60–61-expressing cells and plate on solid medium with sucrose and hygromycin.

• Incubate at 42 °C until colonies are large enough to present a clear GFP signal.

• Using a fluorescence microscope, identify GFP-positive colonies.

• Isolate a GFP-positive colony and grow at 37 °C in liquid medium containing hygromycin until cell clumps are completely dissolved (~ 4 days).

• Streak on solid medium with hygromycin at 37 °C to obtain isolated GFP-positive colonies.

• Pick an isolated colony and grow in liquid medium with hygromycin at 37 °C.

• Verify knockout by PCR and Southern analyses.

• Prepare competent cells and transform with plasmid pML2714.

• Plate on solid media containing kanamycin and grow at 34 °C until colonies are large enough to observe GFP-negative colonies.

• Re-streak GFP-negative colonies on solid medium containing kanamycin and grow at 34 °C.

• To cure the cells of plasmid pML2714, grow at 42 °C in liquid medium without antibiotics until cell clumps are completely dissolved (~ 4 days) and then plate on solid media to obtain isolated colonies.

• Verify curing from plasmid pML2714 and the gfp-hyg cassette by re-streaking on solid media (solid medium with kanamycin and solid medium with hygromycin).

• Choose one candidate that did not grow on kanamycin or hygromycin plates and perform Southern and sequencing analyses to verify successful completion of the gene deletion procedure.