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Regulation of FtsZ Polymer Dynamics by MinC. Construction of pSOT14_ trx-his-minC , pSOT80_ malE-his-minC & pSOT74_ ftsZ-his . Small-scale protein expression, solubility and induction condition test of Trx-His-MinC and MalE-His-MinC.
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Regulation of FtsZ Polymer Dynamics by MinC • Construction of pSOT14_trx-his-minC, pSOT80_malE-his-minC & pSOT74_ftsZ-his. • Small-scale protein expression, solubility and induction condition test of Trx-His-MinC and MalE-His-MinC. • Large-scale protein purification of MalE-His-MinC by nickel column and gel filtration. • Small-scale protein expression, solubility and induction condition test of FtsZ-His. • Large-scale protein purification of FtsZ-His by nickel column and gel filtration. • Observation of FtsZ polymer dynamics under the presence of MalE-His-MinC in vitro by real-time fluorescence microscopy.
Regulation of FtsZ Polymer Dynamics by MinC • Small-scale protein expression, solubility and induction condition test of Trx-His-MinC (~43 kD). Colony 1 Colony 2 TP TPI SUP PE M TP TPI SUP PE 250 150 TP: total protein before induction; TPI: total protein after induction; SUP: supernatant after centrifugation; PE: pellet after centrifugation. 100 75 50 37 25 20 15 kD
Regulation of FtsZ Polymer Dynamics by MinC • Small-scale protein expression, solubility and induction condition test of MalE-His-MinC (~70 kD). Colony 1 Colony 2 TP TPI SUP PE M TP TPI SUP PE 250 150 TP: total protein before induction; TPI: total protein after induction; SUP: supernatant after centrifugation; PE: pellet after centrifugation. 100 75 50 37 25 20 15 kD
Regulation of FtsZ Polymer Dynamics by MinC • Large-scale protein purification of MalE-His-MinC by nickel column. M TP TPP 0 5 10 20 30 40 50 250 250 150 100 75 TP: crude protein extract; TPP: flow through after loading crude extract; No.: equilibration buffer with (No.) mM imidazol. 50 37 25 kD
Regulation of FtsZ Polymer Dynamics by MinC • Large-scale protein purification of MalE-His-MinC by gel filtration. 119 kD* 44 kD* * Estimated by fitting the standard curve of gel filtration column.
Regulation of FtsZ Polymer Dynamics by MinC • Large-scale protein purification of MalE-His-MinC by gel filtration. M 35 37 39 41 43 45 47 49 51 53 55 57 59 250 100 75 50 No.: tube number after gel filtration. 37 25 kD
Regulation of FtsZ Polymer Dynamics by MinC • Construction of pSOT66_gfp-minC. • Construction of pSOT94_ftsZ-cypet. • real-time fluorescence microscopy of Gfp-MinC and FtsZ-CyPet in E. coli.
Future Work • Construction of Plac-ftsZ-mtsftsA, and Plac-ftsZ-mtsminD. • Purification of FtsZ-mtsFtsA, and FtsZ-mtsMinD. • Study on the effect of FtsZ-mts on the liposomes.
