enzymes. In addition, HPLC-MS should be used to identify the
lactonase activity of subsequently expressed proteins [7].- Description of the command-line:
-in Th120.fasta means inputing the Th120.fasta file;
-dbtype prot means this file contains protein not DNA
sequence;
-out Th120DB means creating BLAST database Th12DB. - Description of the command-line:
-query QQenzymes.fasta means inputing the QQenzymes.
fasta file;
-db Th120DB means using the BLAST database Th120DB in
previous step;
-out QQTh120.txt means creating the QQTh120.txt for sav-
ing hits;
-evalue 1e-5 means saving hits with expectation value lower
than 1e^5 ;
-outfmt 6 means results are shown in tabular format;
-max_target_seqs 3 means up to three aligned sequences to
keep. - Many expression vectors with different characteristics can be
used. Actually, expression vectors pET24a (þ) (Novagen) and
pTWIN1 (New England Biolabs) were used in our initial work.
Afterwards, we prefer to use pTWIN1 because this vector can
result with recombinant protein without vector derived amino
acid residues and the CBD tag (chitin binding domain tag) can
be easily removed by altering the pH of elution buffer.
Acknowledgements
Thanks for the permission from Nature Publishing Group and
American Society for Microbiology of reproduction of figures. We
thank Dr. Robert J.C. McLean (Texas State University, USA) for
biosensorA.tumefaciensA136. This work was supported by the
International Science and Technology Cooperation Programme of
China (no. 2012DFG31990), the National Natural Science Foun-
dation of China (no. 41476112), and the Qingdao Postdoctoral
Application Research Project.References
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308 Kaihao Tang and Xiao-Hua Zhang