pDP1381

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	Electrocompetent cells were made from one of these clones, and the bMon14272 bacmid isolated from DH10Bac cells was introduced by electroporation. This final strain, called DE26, was selected on 50 ug/ml kanamycin and 20 ug/ml tetracycline, and serves as the main B2F bacmid production strain.

Protocol tips
Electrocompetent cells were made from one of these clones, and the bMon14272 bacmid isolated from DH10Bac cells was introduced by electroporation. This final strain, called DE26, was selected on 50 ug/ml kanamycin and 20 ug/ml tetracycline, and serves as the main B2F bacmid production strain.

Publication protocol

"In order to construct the blocked attTn7 site strain, a blocking construct was first engineered by introducing the CAT gene encoding chloramphenicol acetyl transferase into an oriR6kγ backbone vector which contains flanking Tn7L and Tn7R sites. The nonessential parts of this vector were removed by a dual EcoRV/HpaI digest, and the CAT gene was amplified by PCR with primers Tn7blockL and Tn7blockR containing EcoRV sites to allow blunt end ligation of the two products. These were ligated using T4 Quick Ligase (New England Biolabs), and transformed into E. coli DE14 which were plated on media containing 100 ug/ml ampicillin and 20 ug/ml chloramphenicol. After overnight incubation, colonies were isolated and grown in liquid media, and plasmid DNA was prepared. Final plasmid DNA was sequence verified using Tn7blockSeq1 and Tn7blockSeq2 primers to ensure the integrity of the CAT-containing insert and the Tn7 transposition sites.

The blocking construct was transformed into E. coli DE24 cells containing the Tn7 transposase helper plasmid (R982-X01) and transformations were plated on 20 ug/ml chloramphenicol after 5 hours of growth to permit transposition to occur. After overnight growth at 37C, colonies were selected and verified for blocking plasmid loss by streaking onto new plates containing 100 ug/ml ampicillin. Clones which did not grow on ampicillin containing media were used to generate genomic DNA preps using the GenElute Genomic DNA kit (Sigma), which were then used for PCR amplification to test for proper insertion of the CAT gene. All clones chosen showed the correct amplification of multiplex PCR products (using primers Tn7genomic1, 2, 3, and 4) showing that transposition occurred into the attTn7 region as expected. PCR products were cleaned using the QiaQuick PCR Purification kit (Qiagen) and were subjected to Sanger sequencing to verify the junctions. As expected, insertion into the attTn7 site occurred at the exact site identified in the literature. Electrocompetent cells were made from one of these clones, and the bMon14272 bacmid isolated from DH10Bac cells was introduced by electroporation. This final strain, called DE26, was selected on 50 ug/ml kanamycin and 20 ug/ml tetracycline, and serves as the main B2F bacmid production strain."

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