- Sequence the whole genome of Th120 in an available company
or institute providing whole genome sequencing service. Gen-
erally, you will get several files including a file containing pre-
dicted genes and a file containing predicted proteins. Rename
the protein sequence file with “Th120.fasta.” - Collect amino acid sequences of all identified AHL enzyme
from literatures [17, 18] in FASTA form and rename the file
with “QQenzymes.fasta.” - The FASTA format is a text-based format and the first line
starts with a “>” and sequence name or other identifiers.
Check the sequence of the AHL lactonase of Th120 in NCBI
database: https://www.ncbi.nlm.nih.gov/protein/
725542033?report¼fasta - Download BLAST software ncbi-blast-2.2.31 þ win32.exe
fromftp://ftp.ncbi.nlm.nih.gov/blast/executables/blastþ/ - Install it in the default Downloads folder. This creates the
directory Downloads\ blast-2.2.31þ. - Copy files of Th120.fasta and QQenzymes.fasta to Downloads\
blast-2.2.31þ\bin folder. - Click on “Start” menu and Type in “cmd” and press “Enter.”
This will bring up the Command Prompt window. - Go into the BLAST folder you just created by entering:
cd Downloads\blast-2.2.31þ\bin - Building a BLAST database with protein sequences of Th120
by entering (seeNote 12):
makeblastdb -in Th120.fasta -dbtype prot -out Th120DB - BLASTP searching putative QQ enzymes using identified QQ
enzymes by entering (seeNote 13):
blastp -query QQenzymes.fasta -db Th120DB -out
QQTh120.txt -evalue 1e-5 -outfmt 6 -max_target_seqs 3 - The QQTh120.txt file will show the three best hits in Th120
with expect value lower than 1 10 ^5 for each identified QQ
enzyme (Table1). - Further confirmation of QQ activities of the four putative QQ
enzymes can be carried out by heterologous overexpression
[16]. In our research, we only found murol 181 and murol
1831 possessing AHL degradative activities, and the AHL
lactonase MomL (murol 1831) was studied in detail [10](see
Note 14).
304 Kaihao Tang and Xiao-Hua Zhang