FastDigest Bsp1407I

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	The retargeted Ll.LtrB targetron (353 bp) was subsequently digested with restriction enzymes HindIII and Bsp1407I (FastDigest, Fermentas) and ligated into pTT_wsfA243 digested with the same restriction enzymes, thereby replacing the wsfA targetron and generating the plasmid pTT_splA101.

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The retargeted Ll.LtrB targetron (353 bp) was subsequently digested with restriction enzymes HindIII and Bsp1407I (FastDigest, Fermentas) and ligated into pTT_wsfA243 digested with the same restriction enzymes, thereby replacing the wsfA targetron and generating the plasmid pTT_splA101.

Publication protocol

"To specifically create a vector for the knockout of splA in P.
larvae 04-309 (ERIC II), we used vector pTT_wsfA243 [42]. This
vector derived from vector pTT_wsfP1176 [49], targeted for
intron insertion at position 243/244 from the start codon of wsfA
in Paenibacillus alvei. WsfA and WsfP both play a role in S-layer
glycosylation reactions in P. alvei [42]. The vector is a fusion of the
commercially available targetron vector pJIR750ai (Sigma)
offering the bacterial mobile group II intron LI.LtrB sequence of
Lactococcus lactis, the Geobacillus-Bacillus-E.coli shuttle vectorpNW33N, and the sgsE S-layer gene promoter of G. stearothermophilus NRS 2004/3a [50] upstream of the intron cassette.
Specific disruption of the splA-gene in P. larvae 04-309 (ERIC II)
was performed following a recently described strategy [49]. The
LI.LtrB targetron of vector pTT_wsfA243 was retargeted prior to
transformation into P. larvae 04-309 (ERIC II). For this purpose,
identification of potential insertion sites of the intron in the splA
open reading frame and design of PCR primers for the
modification of the intron RNA was accomplished by a computer
algorithm (http://www.sigma-genosys.com/targetron). Modified
intron RNA sequences (EBS1, EBS2, d) specifically base pair with
the insertion site target sequences in splA (EBS1, EBS2, d9), leading
to intron insertion and disruption of the gene. The algorithm
predicted 25 possible insertion sites, with position 101 (position
101 from the initial start codon of splA) having the highest
predicted insertion efficiency (E-value 0.004). Three primers (IBS
splA 101, EBS1d splA 101, and EBS2 splA 101, Table 2) were
necessary to retarget the intron to base pair with the splA target
site sequence in the P. larvae chromosome. The retargeted Ll.LtrB
targetron (353 bp) was subsequently digested with restriction
enzymes HindIII and Bsp1407I (FastDigest, Fermentas) and ligated
into pTT_wsfA243 digested with the same restriction enzymes,
thereby replacing the wsfA targetron and generating the plasmid
pTT_splA101. The mutant P. larvae 04-309 strain was analyzed for
the presence of intron DNA in splA and presence/absence of SplA
in 2D-SDS-PA gel electrophoresis as described recently [25]"

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