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PF-MglA(T47A)-V, and pFMglA(Y63A)-V, respectively, except that they confer resistance to nourseothricin. The plasmid pF-MglA has been previously described (10). The pF3 plasmids listed above were generated by replacing the kanamycin resistance gene in pF-MglA-V, pF-MglA, pF-MglA(T47A)-V, and pF-MglA(Y63A)-V with the nourseothricin resistance gene from the previously described pF3 plasmid (21). Genetic screens for mutants of MglA or SspA specifically defective for interaction with PigR. The mglA portion of the fusion in the vector pBR-MglA- was mutagenized using error-prone PCR with Taq polymerase and primers flanking the mglA gene. The PCR product was then digested with the restriction enzymes NdeI and NotI and inserted in the pBR-MglA- vector to generate a library of plasmids that direct the synthesis of MglA- fusion proteins with random mutations in the mglA moiety of the mglA- fusion gene. This library was transformed into KDZif1 Z cells along with plasmids pCL-SspA and pACTR-PigR. Cells were plated on LB containing carbenicillin, spectinomycin, tetracycline, X-Gal (5-bromo-4-chloro-3-indolyl- -D-galactopyranoside; 50 g/ml), IPTG (isopropyl- -D-thiogalactopyranoside; 50 g/ml), and the X-Gal inhibitor tPEG (2-phenylethyl -D-thiogalactoside; 125 g/ml). Approximately 120 colonies were selected in which cells had low levels of lacZ expression compared to cells expressing wild-type MglA- along with PigR-Zif and LVS SspA. These colonies were struck out on LB plates containing carbenicillin to select for only those cells containing the pBRMglA- vector. The pBR-MglA- plasmids expressing various mutant MglA- fusion proteins were then isolated, pooled, and subsequently transformed into KDZif1 Z cells along with pACTR-SspA-Zif. These cells were then plated on LB containing carbenicillin, tetracycline, X-Gal (50 g/ml), and IPTG (50 g/ml). Approximately 40 colonies were selected in which cells expressed levels of lacZ similar to those in cells expressing the wild-type MglA- fusion protein and SspA-Zif. pBRMglA- plasmids were isolated from these colonies and transformed back into KDZif1 Z cells with either pCL-SspA and pACTR-PigR-Zif or pACTR-SspA-Zif and assayed for -galactosidase activity. Plasmids directing the synthesis of MglA- mutant proteins that were specifically defective for interaction with PigR were then sequenced to determine the corresponding mutation. The genetic screen for SspA mutants that were specifically defective for interaction with PigR was essentially the same as that described above forOctober 2014 Volume 196 Numberjb.asm.orgRohlfing and DoveMglA mutants, except that a library of SspA- mutants was generated using error-prone PCR with Taq polymerase and vector pBR-SspA- . This library was analyzed first in KDZif1 Z cells along with the vectors pCL-MglA and pACTR-PigR-Zif. Then, approximately 100 candidate mutants were screened in KDZif1 Z cells containing plasmid pACTRMglA-Zif. Finally, 40 colonies were selected in which cells expressed lacZ levels similar to those in cells expressing the wild-type SspA- fusion protein in the presence of MglA-Zif. Plasmids were isolated from cells from these colonies and tested in the bridge-hybrid assay and two-hybrid assays to confirm that the isolated mutants were specifically defective for interaction with PigR. Plasmids directing the synthesis of SspA- mutant fusion proteins that were specifically defective for interaction with PigR were sequenced to identify the corresponding mutation.
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Bar/tube positions and relative sizes concretethe tomographic photos showin distinctive
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Bar/tube positions and relative sizes concretethe tomographic images showin distinct approaches according to the material; in all instances, it truly is are obtained very accurately the distinctive inclusions frombars/tubes. The bars/tubes inserted inside the possible to appropriately identify no matter the number of the reconstructed tomographic images. concrete the centre of each of the specimens is usually a high-amplitude zone (lowmaterial; in all When it's At modify the ultrasonic image in unique strategies depending on the attenuation). cases, the doable to correctly diametrically opposed, (i.e., when thethe reconstructed tomographic the receiver transducers will not be determine the different inclusions from path in between the emitter and pictures. doesAt the centre of each of the specimens is probes), the received signal is attenuation). Wheneven not cross the central zone in the test a high-amplitude zone (low hugely attenuated the transducers usually are not diametrically opposed, (i.e., when the paththe specimenemitter and than inside the though the path that the ultrasonic signal travels by way of involving the is shorter the receiver will not cross the central case. from the test probes), the received signal is highlyapodizationeven even though diametrically opposed zone This effect is partially corrected by an attenuated procedure, the path that the ultrasonic signal travels through the specimen is shorter than within the diametrically implemented via the inspection method by applying unique gains as a function of the opposed case. This effect is partially corrected by andiametricallyprocedure, implemented via the emitter-receiver positions, (i.e., low gains when apodization opposed, within the limits of inspection technique by variety). distinct gains as a function in the emitter-receiver positions, (i.e., low acquisition dynamic applying gainsIn all the images, the opposed, inside interface (blue zones around the dynamic the test specimens) is when diametrically water-specimen the limits in the acquisition edges of range). In all the images, water-specimen interface (blue zones around the edges of the the aluminium hardly visible since the transmitted pulse is normally hidden by the multiple echoes oftest specimens) is hardlythe ultrasonic tomographic method. Otheroften hidden by the various also observed ring of visible since the transmitted pulse is high attenuation zones are echoes of your aluminium ring to the ultrasonic tomographic technique. are typically positioned close to the surface in the corresponding of natural voids in concrete which Other higher attenuation zones are also observed corresponding to natural voids in concrete which are ordinarily located near the surface of the specimen. specimen.0 inclusion101 inclusion10 20 30 40 50 602 inclusions3 inclusionsPVC30 40 50 60 70 80 20 40 60AluminiumSteelFigure 9. Experimental tomographic reconstruction images with the concrete specimens. specimens.To evaluate the excellent in the ultrasonic reconstructed pictures, a segmented image determined by a To evaluate the high quality with the ultrasonic reconstructed images, a segmented image depending on threshold of the image maximum is generated. The size assigned to the defect depends on the a threshold of the image maximum is generated. The size assigned for the defect depends upon the segmentation threshold. In this case, the criterion applied needs that the reconstructed pictures of segmentation threshold. Within this case, the criterion applied needs that the reconstructed pictures of steel bars, with intermediate attenua.

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Bar/tube positions and relative sizes concretethe tomographic photos showin distinctive Bar/tube positions and relative sizes concretethe tomographic images showin distinct approaches according to the material; in all instances, it truly is are obtained very accurately the distinctive inclusions frombars/tubes. The bars/tubes inserted inside the possible to appropriately identify no matter the number of the reconstructed tomographic images. concrete the centre of each of the specimens is usually a high-amplitude zone (lowmaterial; in all When it's At modify the ultrasonic image in unique strategies depending on the attenuation). cases, the doable to correctly diametrically opposed, (i.e., when thethe reconstructed tomographic the receiver transducers will not be determine the different inclusions from path in between the emitter and pictures. doesAt the centre of each of the specimens is probes), the received signal is attenuation). Wheneven not cross the central zone in the test a high-amplitude zone (low hugely attenuated the transducers usually are not diametrically opposed, (i.e., when the paththe specimenemitter and than inside the though the path that the ultrasonic signal travels by way of involving the is shorter the receiver will not cross the central case. from the test probes), the received signal is highlyapodizationeven even though diametrically opposed zone This effect is partially corrected by an attenuated procedure, the path that the ultrasonic signal travels through the specimen is shorter than within the diametrically implemented via the inspection method by applying unique gains as a function of the opposed case. This effect is partially corrected by andiametricallyprocedure, implemented via the emitter-receiver positions, (i.e., low gains when apodization opposed, within the limits of inspection technique by variety). distinct gains as a function in the emitter-receiver positions, (i.e., low acquisition dynamic applying gainsIn all the images, the opposed, inside interface (blue zones around the dynamic the test specimens) is when diametrically water-specimen the limits in the acquisition edges of range). In all the images, water-specimen interface (blue zones around the edges of the the aluminium hardly visible since the transmitted pulse is normally hidden by the multiple echoes oftest specimens) is hardlythe ultrasonic tomographic method. Otheroften hidden by the various also observed ring of visible since the transmitted pulse is high attenuation zones are echoes of your aluminium ring to the ultrasonic tomographic technique. are typically positioned close to the surface in the corresponding of natural voids in concrete which Other higher attenuation zones are also observed corresponding to natural voids in concrete which are ordinarily located near the surface of the specimen. specimen.0 inclusion101 inclusion10 20 30 40 50 602 inclusions3 inclusionsPVC30 40 50 60 70 80 20 40 60AluminiumSteelFigure 9. Experimental tomographic reconstruction images with the concrete specimens. specimens.To evaluate the excellent in the ultrasonic reconstructed pictures, a segmented image determined by a To evaluate the high quality with the ultrasonic reconstructed images, a segmented image depending on threshold of the image maximum is generated. The size assigned to the defect depends on the a threshold of the image maximum is generated. The size assigned for the defect depends upon the segmentation threshold. In this case, the criterion applied needs that the reconstructed pictures of segmentation threshold. Within this case, the criterion applied needs that the reconstructed pictures of steel bars, with intermediate attenua.

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