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Category:Team Fecal Transplant

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StatusPageUserDate/TimeGO Term (Aspect)ReferenceEvidenceNotesLinks
acceptableVIBVU:G6PIOlsenjay, Team Fecal Transplant2012-04-15 13:33:58 CDTGO:0004347 glucose-6-phosphate isomerase activity (F)PMID:15262231ECO:0000314 direct assay evidence used in manual assertion

Figure 3b: Isomerase activity was readily detectable in PGI-LysAP

from V. vulnificus. Assay of commercially available PGI from rabbit, B. stearothermophilus, and S. cerevisiae also revealed isomerase activity. Determination of the pH optima for isomerase activity for PGI from V. vulnificus.

acceptableBUTFI:GLGBMart1123, Team Fecal Transplant2012-04-15 13:14:01 CDTGO:0003844 1,4-alpha-glucan branching enzyme activity (F)PMID:1938880ECO:0000314 direct assay evidence used in manual assertion

Figure 9 shows the absorbance spectra of the iodine-glucan complex of the a-glucan formed de novo by the purified branching enzyme and phosphorylase a (ratio of 1:1). The combination of the actions of phosphorylase a and GlgB yielded a branched product with a spectrum similar to glycogen. The initial Xmax was 490nm, but as the reaction proceeded decreased to 430nm, indicating a highly branched product.

requireschanges9ASCO:F1T2J9Olsenjay, Team Fecal Transplant2012-04-15 11:20:48 CDTGO:0016616 oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor (F)PMID:21672638ECO:0000314 direct assay evidence used in manual assertion

Figure 1: Time dependent changes in PLA levels were monitored using HPLC. The PLA concentration linearly increased (Fig. 1A), indicating that the strain produced PLA at a constant rate. Adding PPA, a potential PLA substrate, to the culture medium increased the amount of PLA produced by strain TK1 (Fig. 1B). After 48-h of culture, 1.5 mM PPA was consumed and 0.8 mM of PLA accumulated in the medium,indicating that 53% of the PPA was converted to PLA. When cultured in MM containing phenylalanine, strain TK1 consumed phenylalanine and produced PLA (Fig. 1C). After the initial 24-h of incubation, 5.0 mMphenylalanine was consumed and 5.7 mMPLA was generated,indicating that most of the added phenylalanine was converted to PLA. We detected phenylalanine aminotransferase (PAT) (1.9±0.2 μmol min−1 mg−1) and PPR (see below) activities in the fungal cell-free extracts.

These results together with the higher levels of PLA

being generated from either PPA or phenylalanine than from glucose when added as a carbon source, indicated that the fungus generates PLA via PPA and/or phenylalanine (Fig. 1D), and that biosynthesized PPA limits PLA production from glucose.

acceptableESCVU:CRTXOlsenjay, Team Fecal Transplant2012-04-15 10:37:53 CDTGO:0046527 glucosyltransferase activity (F)PMID:1409639ECO:0000314 direct assay evidence used in manual assertion

Figure 3: Extracts from plasmid-containing Es. coli strains were

assayed for in vitro glucosyltransferase activity by incubation with zeaxanthin and UDP-[14C]glucose. When an extract from induced Es. coli BL21(DE3) cells containing pAPUX was used, significant amounts of the yellow-colored radiolabeled products, zeaxanthin mono- and diglucosides, were observed. As a negative control, an extract from Es. coli BL21(DE3) cells containing the pET-3b vector without crtX was incubated with zeaxanthin and UDP-[14C]glucose in the in vitro assay, and no product was observed. Thin layer chromatographic analysis of the products of this assay is shown in the figure.

acceptableDEIRA:Q9RW08Olsenjay, Team Fecal Transplant2012-04-15 07:06:29 CDTGO:0016491 oxidoreductase activity (F)PMID:17464079ECO:0000314 direct assay evidence used in manual assertion

Figure 5: Purified DR0861 protein from D. radioduran converted 15,15'-cis-phytoene to lycopene detected by HPLC. This confirmed that the lycopene synthesized from phytoene in D. radioduran was catalyzed by the DR0681 protein.

requireschangesCITSI:Q84X90Olsenjay, Team Fecal Transplant2012-04-14 12:02:19 CDTGO:0046905 phytoene synthase activity (F)PMID:19813015ECO:0000314 direct assay evidence used in manual assertion

Figure 3(a),(b),(c):HPLC analysis revealed that the

GGPP-accumulating E. coli cells transformed with the CsPSY construct showed a peak (Fig. 3b, peak 1) that was not detected in the empty vector control, pACCRT-E/pET-28a (Fig. 3a). The retention time and absorption spectrum of the peak 1 matched well with those of the peak 2 presented in the positive control carrying pACCRT-EB for phytoene biosynthesis (Fig. 3c, peak 2).

acceptableLEUCI:D5FS20Olsenjay, Team Fecal Transplant2012-04-13 20:27:44 CDTGO:0047849 dextransucrase activity (F)PMID:21229247ECO:0000314 direct assay evidence used in manual assertion

Figure 2: Highperformance

anion-exchange chromatography with pulsed amperometric detection was used to compare profiles. L. citreum B/110-1-2 (WT dsrF), DSR-F-DSP-DGBD (recombinant dextransucrase),DSR-S from mesenteroides NRRLB-512F(DSR-S is a dextransucrase). L. citreum B/110-1-2 produces a series of isomaltodextrins analogs with retention times that were very similar to the ones produced by the dextransucrase DSR-S from L. mesenteroides NRRLB-512F.

requireschangesCAMJE:ARLYOlsenjay, Team Fecal Transplant2012-04-13 16:33:30 CDTGO:0004056 argininosuccinate lyase activity (F)PMID:11683360ECO:0000314 direct assay evidence used in manual assertion

E. coli C600 (WT functional argH),E. coli W3678 (no argH gene), C. jejuni TGH9011 (WT functional argH), and transformed E. coli (C. jejuni TGH9011 argH gene) were examined for ASL activity. E. coli C600, which is wild type

with respect to the argH gene, showed a specific activity of 1.1 + 0.17 ,umol of arginine per h per mg of protein. C. jejuni TGH9011 showed a specific activity of 1.3 ± 0.32 ,umol of arginine per h per mg of protein. E. coli W3678 did not possess any significant ASL activity compared with extract-free controls. The transformed strain pARGH1-1 had a specific activity of 5.7 ± 1.2 ,imol of arginine per h per mg of protein. This result confirms that the protein derived from the recombinant plasmid possesses argininosuccinate lyase activity activity.

acceptableXANCP:AOTCOlsenjay, Team Fecal Transplant2012-04-13 15:54:26 CDTGO:0043857 N-acetylornithine carbamoyltransferase activity (F)PMID:16585758ECO:0000314 direct assay evidence used in manual assertion

Table 1: Purified ArgF′ enzyme from Xanthomonas catalyzed the AOTCase reaction which forms N-acetylcitrulline from N-acetylornithine and carbamyl phosphate.

Figure 4: LC-MS analysis of enzymatically synthesized N-acetylcitrulline. The enzymatic product of the X. campestris AOTCase reaction has the same mass ion and retention time as chemically synthesized N-acetylcitrulline.

acceptableRHOMR:MGSMart1123, Team Fecal Transplant2012-04-12 19:47:35 CDTGO:0051479 mannosylglycerate biosynthetic process (P)PMID:10585410ECO:0000314 direct assay evidence used in manual assertion

Table 2 shows the activity of the native and recombinant enzymes and their ability to utilize substrates in the mannosylglycerate biosynthetic pathway. The native enzyme was obtained by electroelution and had a specific activity of 70mmol/min/mg of protein. The substrates used to determine the specificity of Mgs were GDP mannose, ADP mannose, UDP mannose,mannose 1-phosphate, and mannose 6-phosphate as sugar donors,and D-glycerate,L-glycerate, and D-3-P-glycerate as sugar acceptors. GDP glucose, UDP glucose, and ADP glucose were tested as

sugar donors, and glycerol 3-phosphate, glucose 6-phosphate,glucose 1-phosphate, and glucose as sugar acceptors in an effort to also identify the ability of Mgs to catalyze the synthesis of other glycosylconjugates. Mgs was specific for GDP mannose and D-glycerate and had an absolute stereospecificity for D-glycerate. UDP mannose and GDP mannose were the only sugar nucleotides used by the enzyme. UDP mannose was used very little, less than 3% of that of GDP mannose. NaCl and KCl (50-500 mM concentrations) had no effect on enzyme activity. However, Mg2+ was required for optimal activity shown by the 2.5-fold lower activity in absence of the cation.

requireschangesRHOMR:O52754Mart1123, Team Fecal Transplant2012-04-12 18:25:31 CDTGO:0004553 hydrolase activity, hydrolyzing O-glycosyl compounds (F)PMID:9799108ECO:0000314 direct assay evidence used in manual assertion

Figures 5 and 6 confirm the hydrolase function of the LamR enzyme, which is expressed by the lamR gene. Both figures show digests separated by TLC. In figure 5, the intermediate appearance of oligosaccharides detected after a short incubation time prove that the hydrolysis of 1,3-1,4-beta-glucan and laminarin follows the endo type action pattern.

In figure 6, LamR displayed a weak and significant activity against cellobiosyl-glucose G1-4G1-3Gr, which suggests that LamR is able to cut specific beta linkages only in low-molecular-mass carbohydrates.

requireschangesRHOMR:Q66SU9Mart1123, Team Fecal Transplant2012-04-12 12:02:06 CDTGO:0008843 endochitinase activity (F)PMID:15583965ECO:0000314 direct assay evidence used in manual assertion

Figure 6 shows changes in viscosity from the DDA 73% chitosan. This clearly indicates that hydrolysis occurred inside the polymers, proving that Chi is an endoacting chitinase on chitopolysaccharides.

acceptableRHOMR:O33897Mart1123, Team Fecal Transplant2012-04-12 11:37:59 CDTGO:0008810 cellulase activity (F)PMID:9581291ECO:0000314 direct assay evidence used in manual assertion

Figures 2,3, and 4 show SDS page gels for the native CelA enzyme, the recombinant form of the enzyme, and a gel comparing the activity and protein of both. Figure 2 shows the native CelA molecular mass corresponds to the expected value. This information is important for future comparisons to recombinant types. Figure 3 shows the recombinant CelA molecular mass and activity after boiling with proteinase K. The control (lane 2) showed activity bands at higher molecular weights than the native enzyme. After boiling, activity began to disappear and a protein band of 26 kDa appeared in lanes 3, 4, and 5. Figure 4 shows an SDS gel with CMC for both enzymes. The native activity band appeared in a corresponding position to the smallest activity band of the recombinant enzyme. The same activity bands were seen regardless of heating with protease.

acceptableDEIRA:Y079Olsenjay, Team Fecal Transplant2012-04-10 21:44:09 CDTGO:0016787 hydrolase activity (F)PMID:18512963ECO:0000314 direct assay evidence used in manual assertion

Figure 5: NMR spectroscopy was used to follow the

phosphorus resonances for three substrates, CDP, IDP, and CTP, alone and with the addition of DP_0079. This showed the hydrolase activity of DP_0079 by observing the hydrolase products CMP + Pi, IMP + Pi, and CMP + PPi only with the addition of DP_0079.


MSU Internal Competition

StatusPageUserDate/TimeGO Term (Aspect)ReferenceEvidenceNotesLinks
requireschangesECOLI:GLNPOlsenjay, Team Fecal Transplant2012-04-01 11:30:17 CDTGO:0006810 transport (P)PMID:3542962ECO:0000315 mutant phenotype evidence used in manual assertion

Figure 3: Transport assay was done of E.coli parental strain PSM2 with a functional glnP and of a glnP mutant strain PSM221. PSM221 with the mutant glnp showed a greatly reduced ability to transport glutamine compared to with PSM2 with the functional glnP which had a much greater ability to transport glutamine.

requireschangesSTRPN:A7KT31Mart1123, Team Fecal Transplant2012-03-29 12:38:22 CDTGO:0086080 protein binding involved in heterotypic cell-cell adhesion (F)PMID:17850254ECO:0000315 mutant phenotype evidence used in manual assertion

Figure 5 RrgA is involved in pilus-mediated colonization of the upper respiratory tract in mice. The mice were infected with T4 phage with rrgA and rrgB/rrgC deletions respectively. Seven days after infection, Mice infected with T4 rrgA deletion phage had significantly less bacteria in the nasopharynx than mice infected with T4 rrgB/rrgC deletion phage. This demonstrates that rrgA is important for pilli assembly and strong cell wall adhesion.


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