Southern blot analysis was performed to determine whether the shuttle vectors (pFJ1, pFJ4, and pFJ6) had integrated into the J7-F chromosome and to measure their copy numbers.
To construct shuttle vectors for J7 derivatives, oriCs in the J7 chromosome were predicted using the web-based Ori-Finder 2 tool.
To assess the structural stability of all shuttle vectors (pFJ1, pFJ3, pFJ4, pFJ5, pFJ6, and pFJ7) and verify their ability to replicate independently in J7-F cells, plasmid DNA was extracted from each vector transformant and back-transformed into E.
Shuttle vectors containing chromosome oriCs are usually present at one copy per chromosome .
Compatible shuttle vectors are excellent tools for investigating protein-protein and protein-DNA interactions in prokaryotes.
Second, the molecular weights of the vectors constructed here were all around 6 kb, which is much smaller than that of most of the archaeal shuttle vectors previously reported , including pYCJ (about 9.9 kb).
Six of the shuttle vectors containing one or two of the oriCs could replicate independently in J7-F cells, suggesting that at least six of the 12 predicted oriCs had the ability to initiate DNA replication.
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