Shane+A.

= = =Week 15:=

Gen analysis was completed. Enzyme assay was also completed.

CB-kinUT was also screened. Results are as posted:


 * Score || S(PLP) || S(hbond) || S(cho) || S(metal) || DE(clash) || DE(tors) || intcor || time || File name |||| Ligand name ||  ||   ||
 * 88.13 || -86.27 || 1.25 || 0 || 0 || 0 || 2.9 || 3.93 || 38.301 || 'output_301_400/gold_soln_PTPBvsCBkinUTRun1_m330_1.sdf' |||||| '7952001|CB-kin_UT|sdf|3594|dock1' ||  ||
 * 85.51 || -81.5 || 1.58 || 0 || 0 || 4.65 || 1.5 || 6.63 || 28.837 || 'output_201_300/gold_soln_PTPBvsCBkinUTRun1_m281_1.sdf' |||||| '7914191|CB-kin_UT|sdf|2986|dock9' ||  ||
 * 83.59 || -79.7 || 1.59 || 0 || 0 || 0 || 1.31 || 1.73 || 18.537 || 'output_101_200/gold_soln_PTPBvsCBkinUTRun1_m121_10.sdf |||||||| ' '7702584|CB-kin_UT|sdf|1547|dock1' ||
 * 83.58 || -79.46 || 1.96 || 0 || 0 || 0 || 2.78 || 3.81 || 30.406 || 'output_201_300/gold_soln_PTPBvsCBkinUTRun1_m282_5.sdf' |||||| '7909619|CB-kin_UT|sdf|2836|dock6' ||  ||
 * 83.22 || -85.93 || 0 || 0 || 0 || 0.46 || 2.24 || 2.23 || 29.963 || 'output_101_200/gold_soln_PTPBvsCBkinUTRun1_m161_7.sdf' |||||| '7765175|CB-kin_UT|sdf|1935|dock5' ||  ||
 * 83.08 || -84.95 || 0 || 0 || 0 || 3 || 2.18 || 5.49 || 23.819 || 'output_301_400/gold_soln_PTPBvsCBkinUTRun1_m361_2.sdf' |||||| '7961087|CB-kin_UT|sdf|3736|dock2' ||  ||
 * 82.99 || -78.93 || 1.54 || 0 || 0 || 0 || 1.15 || 1.72 || 15.531 || 'output_201_300/gold_soln_PTPBvsCBkinUTRun1_m241_6.sdf' |||||| '7851616|CB-kin_UT|sdf|2421|dock5' ||  ||
 * 82.79 || -80.42 || 1.47 || 0 || 0 || 0 || 2.09 || 2.15 || 9.578 || 'output_201_300/gold_soln_PTPBvsCBkinUTRun1_m246_3.sdf' |||||| '7856892|CB-kin_UT|sdf|2457|dock1' ||  ||
 * 82.2 || -80.85 || 2 || 0 || 0 || 3.01 || 3.55 || 4.89 || 13.122 || 'output_301_400/gold_soln_PTPBvsCBkinUTRun1_m313_1.sdf' |||||||| '7914340|CB-kin_UT|sdf|2996|dock10' ||
 * 81.98 || -76.63 || 2.07 || 0 || 0 || 0 || 1.56 || 2.23 || 7.496 || 'output_201_300/gold_soln_PTPBvsCBkinUTRun1_m201_1.sdf' |||||||| '7844209|CB-kin_UT|sdf|2331|dock10' ||
 * 81.8 || -76.13 || 2.96 || 0 || 0 || 0 || 2.09 || 0.97 || 30.324 || 'output_101_200/gold_soln_PTPBvsCBkinUTRun1_m166_9.sdf' |||||| '7728798|CB-kin_UT|sdf|1631|dock6' ||  ||
 * 81.71 || -77.22 || 1 || 1 || 0 || 0 || 0.78 || 0.06 || 26.065 || 'output_101_200/gold_soln_PTPBvsCBkinUTRun1_m177_6.sdf' |||||| '7740896|CB-kin_UT|sdf|1711|dock3' ||  ||
 * 81.63 || -75.95 || 2.71 || 0 || 0 || 0 || 1.9 || 1.09 || 14.801 || 'output_101_200/gold_soln_PTPBvsCBkinUTRun1_m164_3.sdf' |||||| '7725753|CB-kin_UT|sdf|1607|dock8' ||  ||
 * 81.44 || -78.17 || 1.56 || 0 || 0 || 0 || 3.06 || 4.67 || 30.194 || 'output_301_400/gold_soln_PTPBvsCBkinUTRun1_m327_2.sdf' |||||| '7929362|CB-kin_UT|sdf|3324|dock1' ||  ||
 * 81.06 || -78.92 || 1 || 0 || 0 || 0.6 || 3.14 || 6.02 || 16.735 || 'output_201_300/gold_soln_PTPBvsCBkinUTRun1_m242_6.sdf' |||||| '7876071|CB-kin_UT|sdf|2553|dock4' ||  ||
 * 80.83 || -78.46 || 1.68 || 0 || 0 || 0 || 2.33 || 1.98 || 14.959 || 'output_301_400/gold_soln_PTPBvsCBkinUTRun1_m321_2.sdf' |||||| '7931547|CB-kin_UT|sdf|3369|dock6' ||  ||
 * 80.58 || -82.98 || 0 || 0 || 0 || 1.17 || 0.66 || 0.11 || 32.938 || 'output_201_300/gold_soln_PTPBvsCBkinUTRun1_m290_10.sdf' |||||| '7916999|CB-kin_UT|sdf|3031|dock8' ||  ||
 * 80.44 || -78.94 || 1 || 0 || 0 || 0.01 || 3.55 || 5.62 || 29.826 || 'output_101_200/gold_soln_PTPBvsCBkinUTRun1_m126_10.sdf |||||||| ' '7666127|CB-kin_UT|sdf|1314|dock10' ||
 * 80.35 || -75.75 || 2 || 0 || 0 || 0 || 1.21 || 0.93 || 30.908 || 'output_101_200/gold_soln_PTPBvsCBkinUTRun1_m162_4.sdf' |||||||| '7734334|CB-kin_UT|sdf|1672|dock10' ||
 * 80.05 || -80.22 || 1 || 0 || 0 || 0.04 || 2.22 || 0.93 || 25.7 || 'output_201_300/gold_soln_PTPBvsCBkinUTRun1_m297_1.sdf' |||||| '7922564|CB-kin_UT|sdf|3174|dock5' ||  ||
 * 80.05 || -80.22 || 1 || 0 || 0 || 0.04 || 2.22 || 0.93 || 25.7 || 'output_201_300/gold_soln_PTPBvsCBkinUTRun1_m297_1.sdf' |||||| '7922564|CB-kin_UT|sdf|3174|dock5' ||  ||

Fig. 1: Top 20 compounds with top scores from GOLD. Screening of PTPB.

Fig. 2: Enzyme Assay results. The largest absorbance reading was at an enzyme concentration of 0.63. , =Week 14:=

Virtual Screening was edited and re run on the CB306 library. Purification protocol was also completed.



Fig. 3: Top 20 ligands from Virtual Screening in GOLD for the target protein
 * # Score S(PLP) S(hbond) S(cho) S(metal) DE(clash) DE(tors) intcor time File name Ligand name ||
 * 73.07 || -69.16 || 2 || 0 || 0 || 0 || 1.27 || 0.46 || 15.728 || 'output_103_204/gold_soln_cb_306_3d_m127_2.sdf' || '6956655' ||
 * 66.66 || -65.75 || 0.43 || 0 || 0 || 0 || 0.57 || 0.67 || 31.062 || 'output_103_204/gold_soln_cb_306_3d_m145_2.sdf' || '7558664' ||
 * 66.15 || -62.01 || 1.43 || 0 || 0 || 0 || 0.13 || 0.1 || 13.072 || 'output_205_306/gold_soln_cb_306_3d_m209_3.sdf' || '7766330' ||
 * 64.82 || -65.2 || 1 || 0 || 0 || 0 || 2.19 || 1 || 28.698 || 'output_1_102/gold_soln_cb_306_3d_m83_8.sdf' || '5627579' ||
 * 64.74 || -59.09 || 1.99 || 0 || 0 || 0 || 0.19 || 0.04 || 16.246 || 'output_103_204/gold_soln_cb_306_3d_m203_2.sdf' || '7748043' ||
 * 64.65 || -59.65 || 1.98 || 0 || 0 || 0 || 0.49 || 0.03 || 27.589 || 'output_103_204/gold_soln_cb_306_3d_m201_4.sdf' || '7746693' ||
 * 64.57 || -63.23 || 1 || 0 || 0 || 0 || 0.85 || 0.04 || 19.294 || 'output_103_204/gold_soln_cb_306_3d_m137_9.sdf' || '7493042' ||
 * 64.52 || -62.1 || 0.99 || 0.32 || 0 || 0 || 0.8 || 0.05 || 21.003 || 'output_103_204/gold_soln_cb_306_3d_m184_4.sdf' || '7722615' ||
 * 64.44 || -63.16 || 1.28 || 0 || 0 || 0 || 1.32 || 0.1 || 23.602 || 'output_103_204/gold_soln_cb_306_3d_m135_5.sdf' || '7474459' ||
 * 64.42 || -68.41 || 0 || 0 || 0 || 0 || 2.08 || 0.17 || 39.292 || 'output_1_102/gold_soln_cb_306_3d_m85_9.sdf' || '5654317' ||
 * 64.02 || -61.77 || 1 || 0 || 0 || 0 || 1.45 || 2.15 || 18.634 || 'output_205_306/gold_soln_cb_306_3d_m206_8.sdf' || '7757183' ||
 * 63.75 || -57.28 || 2.39 || 0 || 0 || 0 || 0.35 || 0.01 || 6.431 || 'output_1_102/gold_soln_cb_306_3d_m20_2.sdf' || '5158497' ||
 * 63.43 || -61.4 || 1 || 0 || 0 || 0 || 0.6 || 0.04 || 8.151 || 'output_103_204/gold_soln_cb_306_3d_m158_2.sdf' || '7637949' ||
 * 63.35 || -64.42 || 0 || 0 || 0 || 0 || 0.62 || 0.04 || 9.427 || 'output_205_306/gold_soln_cb_306_3d_m276_1.sdf' || '9027311' ||
 * 63.09 || -60.8 || 0.89 || 0 || 0 || 0 || 0.22 || 0.03 || 6.5 || 'output_205_306/gold_soln_cb_306_3d_m227_1.sdf' || '7870889' ||
 * 62.81 || -63.73 || 1 || 0 || 0 || 0 || 1.98 || 0.04 || 21.686 || 'output_103_204/gold_soln_cb_306_3d_m143_1.sdf' || '7543758' ||
 * 62.5 || -58.88 || 1.76 || 0 || 0 || 0 || 0.85 || 0.06 || 29.767 || 'output_205_306/gold_soln_cb_306_3d_m223_10.sdf' || '7848095' ||
 * 62.33 || -60.59 || 0.99 || 0 || 0 || 0 || 0.68 || 0.02 || 5.607 || 'output_205_306/gold_soln_cb_306_3d_m221_1.sdf' || '7829885' ||
 * 62.31 || -52.25 || 4.02 || 0 || 0 || 0 || 1.13 || 0.12 || 27.75 || 'output_103_204/gold_soln_cb_306_3d_m187_1.sdf' || '7726180' ||
 * 62.11 || -60.32 || 1 || 0 || 0 || 0 || 0.64 || 0.07 || 6.909 || 'output_103_204/gold_soln_cb_306_3d_m157_1.sdf' || '7635462' ||
 * 62.11 || -60.32 || 1 || 0 || 0 || 0 || 0.64 || 0.07 || 6.909 || 'output_103_204/gold_soln_cb_306_3d_m157_1.sdf' || '7635462' ||

=Week 13:= Good, but double check these scores - you might have a column transposed - Dr. B 11/26/12 Fig 1: Top 21 ligands from CBkinUT binding to the protein PTPB. Scores are negative. Least negative score is the best.
 * Score || S(PLP || ) S(hbond) || S(cho) || S(metal) || DE(clash) || DE(tors) || intcor || time || File name |||| Ligand name ||  ||
 * -46.24 || 49.09 || 1.81 || 0 || 0 || 0 || 1.33 || 0.08 || 27.961 || 'output_1_100/gold_soln_PTPBvsCBkinUT_Run1_m81_7.sdf' |||||| '7651891|CB-kin_UT|sdf|1208|dock2' ||
 * -50.51 || 55.42 || 2.09 || 0 || 0 || 0.04 || 0.67 || 0.01 || 7.351 || 'output_101_200/gold_soln_PTPBvsCBkinUT_Run1_m161_2.sdf' |||||| '7741517|CB-kin_UT|sdf|1716|dock8' ||
 * -50.9 || 57.43 || 2.81 || 0 || 0 || 0 || 0.99 || 0.05 || 27.641 || 'output_301_400/gold_soln_PTPBvsCBkinUT_Run1_m321_10.sdf' |||||| '7981885|CB-kin_UT|sdf|3924|dock2' ||
 * -54.38 || 52.33 || 0.07 || 0 || 0 || 0 || 1.15 || 0.02 || 18.202 || 'output_301_400/gold_soln_PTPBvsCBkinUT_Run1_m322_7.sdf' |||||| '7930669|CB-kin_UT|sdf|3351|dock9' ||
 * -57.52 || 58.42 || 1 || 0 || 0 || 0 || 1.06 || 0.01 || 5.612 || 'output_1_100/gold_soln_PTPBvsCBkinUT_Run1_m1_3.sdf' |||||| '6610904|CB-kin_UT|sdf|337|dock5' ||
 * -60.22 || 57.75 || 0 || 0 || 0 || 0 || 1.36 || 0.13 || 33.259 || 'output_1_100/gold_soln_PTPBvsCBkinUT_Run1_m20_4.sdf' |||||| '7354819|CB-kin_UT|sdf|574|dock2' ||
 * -60.82 || 58.06 || 0.26 || 0.17 || 0 || 0 || 2.04 || 0.03 || 9.515 || 'output_201_300/gold_soln_PTPBvsCBkinUT_Run1_m241_1.sdf' |||||| '7882539|CB-kin_UT|sdf|2580|dock10' ||
 * -65.33 || 65.68 || 0.51 || 0.23 || 0 || 0 || 1.04 || 0.21 || 33.427 || 'output_1_100/gold_soln_PTPBvsCBkinUT_Run1_m82_7.sdf' |||||| '7720858|CB-kin_UT|sdf|1567|dock6' ||
 * -68.92 || 73 || 2.16 || 0 || 0 || 0 || 1.27 || 0.15 || 41.019 || 'output_1_100/gold_soln_PTPBvsCBkinUT_Run1_m3_6.sdf' |||||| '7336443|CB-kin_UT|sdf|569|dock1' ||
 * -69.13 || 70.44 || 1.81 || 0 || 0 || 0 || 2.09 || 0.03 || 5.562 || 'output_1_100/gold_soln_PTPBvsCBkinUT_Run1_m2_1.sdf' |||||| '6590610|CB-kin_UT|sdf|329|dock1' ||
 * -69.22 || 68.94 || 0.05 || 0 || 0 || 0 || 0.22 || 0.01 || 19.037 || 'output_101_200/gold_soln_PTPBvsCBkinUT_Run1_m166_5.sdf' |||||| '7835411|CB-kin_UT|sdf|2309|dock3' ||
 * -69.65 || 72.02 || 1.35 || 0 || 0 || 0 || 0.86 || 0.04 || 25.169 || 'output_301_400/gold_soln_PTPBvsCBkinUT_Run1_m324_8.sdf' |||||| '7972328|CB-kin_UT|sdf|3859|dock10' ||
 * -73.19 || 78.34 || 2.4 || 0 || 0 || 0 || 1.18 || 0.29 || 9.193 || 'output_1_100/gold_soln_PTPBvsCBkinUT_Run1_m84_3.sdf' |||||| '7723085|CB-kin_UT|sdf|1580|dock4' ||
 * -76.28 || 78 || 0.98 || 0.3 || 0 || 0 || 1.29 || 0.08 || 32.742 || 'output_301_400/gold_soln_PTPBvsCBkinUT_Run1_m323_1.sdf' |||||| '7988034|CB-kin_UT|sdf|3966|dock10' ||
 * -76.37 || 77.71 || 1.02 || 0 || 0 || 0 || 1.4 || 0.84 || 10.145 || 'output_1_100/gold_soln_PTPBvsCBkinUT_Run1_m83_1.sdf' |||||| '7648767|CB-kin_UT|sdf|1182|dock3' ||
 * -76.46 || 77.92 || 1.78 || 0 || 0 || 0 || 1.98 || 0.1 || 9.818 || 'output_101_200/gold_soln_PTPBvsCBkinUT_Run1_m168_3.sdf' |||||| '7806374|CB-kin_UT|sdf|2209|dock8' ||
 * -76.93 || 75.27 || 0 || 0 || 0 || 0 || 1.24 || 0.82 || 21.077 || 'output_101_200/gold_soln_PTPBvsCBkinUT_Run1_m109_3.sdf' |||||| '7658051|CB-kin_UT|sdf|1261|dock3' ||
 * -77.05 || 78.22 || 0.77 || 0 || 0 || 0 || 1.08 || 1.02 || 5.613 || 'output_1_100/gold_soln_PTPBvsCBkinUT_Run1_m85_2.sdf' |||||| '7701439|CB-kin_UT|sdf|1542|dock1' ||
 * -77.65 || 82.38 || 1.99 || 0 || 0 || 0 || 0.63 || 0.02 || 7.612 || 'output_1_100/gold_soln_PTPBvsCBkinUT_Run1_m87_4.sdf' |||||| '7696822|CB-kin_UT|sdf|1520|dock2' ||
 * -78.74 || 78.2 || 0.78 || 0 || 0 || 0 || 1.92 || 0.95 || 5.933 || 'output_1_100/gold_soln_PTPBvsCBkinUT_Run1_m86_2.sdf' |||||| '7670225|CB-kin_UT|sdf|1338|dock2' ||
 * -79.2 || 77.23 || 1 || 0 || 0 || 2.75 || 1.16 || 0.08 || 10.297 || 'output_101_200/gold_soln_PTPBvsCBkinUT_Run1_m162_4.sdf' |||||| '7754931|CB-kin_UT|sdf|1866|dock5' ||
 * -79.2 || 77.23 || 1 || 0 || 0 || 2.75 || 1.16 || 0.08 || 10.297 || 'output_101_200/gold_soln_PTPBvsCBkinUT_Run1_m162_4.sdf' |||||| '7754931|CB-kin_UT|sdf|1866|dock5' ||

Surrogate cloning was also completed with Joey's protein in mind. Bacteria cells grown were also sonicated. Next week, the protein will be harvested and a protein gel check will be used to confirm the correct protein was found and that it had not dimerized.

=Week 12:= Good - Dr. B 11/19/12





=Week 11:=

After a few attempts to get the PNIC-Bsa4 at a high concentration, cloning was attempted. Only one colony of the bacteria grew after one day, but I will try cloning again to grow more colonies. Fig. 1: DH5alpha bacteria with gene of interest plated with Kanamycin and 5% sucrose.

Fig. 2: GEL check of pNIC-Bsa4 cut with T4 polymerase =Week 10:=





This week I performed several MIDI preps for the pNIC-Bsa4 to yield a higher concentration in order to continue the cloning process. However, my first MIDI prep resulted in a low concentration due to someone switching the caps on the elution and wash buffer solution. My next MIDI prep resulted in a decent yield, so next week I will try to finish the cloning process using the 69.4 ng/uL concentration of pNIC-Bsa4. I also completed a PCR cleanup on the pNIC-Bsa4, but that only boosted my concentration by 15 ng/uL.

=Week 9:=



This low concentration shows that the procedure needs to be redone.

Since there were no large concentrations of the pNIC-Bsa4, new preparation using MIDI prep has to be completed. This will hopefully increase the concentration of the cut pNIC-Bsa4. From there, I can continue the cloning process.

=Week 8:= 102112 - good -- Dr. B Virtual Screening Refresher was completed:


 * Fitness || S(hb_ext) || S(vdw_ext) || S(hb_int) || S(int) |||| Ligand name ||  ||
 * 81.65 || 5.62 || 60.65 || 0 || -7.36 |||||| '9039798|CB5k_10|sdf|947|dock8' ||
 * 81.56 || 6 || 54.08 || 0 || 1.21 |||||| '9040519|CB5k_10|sdf|1056|dock6' ||
 * 81.26 || 13.11 || 53.2 || 0 || -5.01 |||||| '9036564|CB5k_10|sdf|426|dock10' ||
 * 81.26 || 4.54 || 59.33 || 0 || -4.85 |||||| '9057940|CB5k_10|sdf|3998|dock2' ||
 * 80.84 || 18.09 || 51.04 || 0 || -7.43 |||||| '9038483|CB5k_10|sdf|723|dock6' ||
 * 80.3 || 6.06 || 61.95 || 0 || -10.94 |||||| '9037146|CB5k_10|sdf|529|dock1' ||
 * 79.78 || 17.98 || 48.95 || 0 || -5.50 |||||| '9035520|CB5k_10|sdf|251|dock7' ||
 * 77.42 || 12 || 49.84 || 0 || -3.12 |||||| '9059625|CB5k_10|sdf|4116|dock10' ||
 * 77.1 || 1.74 || 55.37 || 0 || -0.78 |||||| '9038770|CB5k_10|sdf|778|dock3' ||
 * 76.89 || 12 || 54.62 || 0 || -10.21 |||||| '9049544|CB5k_10|sdf|2822|dock4' ||
 * 76.89 || 12 || 54.62 || 0 || -10.21 |||||| '9049544|CB5k_10|sdf|2822|dock4' ||

Table 1: Table of Top 10 Ligands through the use of GOLD.

Cloning was also started. Will be finished next week. I had to improve my PCR^2 concentration. PCR was redone. Gel will be completed next week as well.

=Week 7:= 101612 - Shane, go ahead and try cloning - but also start working on making more PCR^2 so that you can get a higher Nanodrop conc of your gene. Good job on the PCR's though - Dr. B

Virtual Screening Refresher was also started. The first overnight run was submitted. Second run will be submitted and analysis will be complete by Tuesday morning.



After the PCR cleanup, the concentration of my target resulted in 18 ng/uL. This is slightly low, and in order to make up for this, I will add an extra volume. The 260/280 value was 1.69 and my 260/230 value was .94.



Lane 1: 100 bp ladder Lane 2-5: My PCR^2 mix

This gel electrophoresis shows that my target was made and had successfully gone through the PCR reaction. Thus, all my bands match up at the designated area.

=Week 6:=



Lane 1: 100 bp ladder Lane 2: My PCR mix Lane 3: Mihir's PCR mix Lane 4: Akhilesh's PCR mix

The mix consisted of a reaction buffer, Hod Start Polymerase, MgSO4, 1 ul of a mix of primers (22 primers LmP, 78 ul of nanopure water), and dNTP.

My PCR mix on the gel showed up as a variety of bands. This is probably due to the primers forming the heavier strands on the top and not completing the bands on the bottom.

=Week 5: Sept. 24-28 = 100912 - Shane - do you have your updated gels?

Our secondary PCR failed.



10012 - after you do PCR #2, you can move ahead to cloning. Also, show your Primers for primer design. I don't know what to order here. -- Dr. B --why is there a * star in your FASTA sequence here?

VIRTUAL PLASMID FOR TARGET: Design Primer Protocl Results:

Upstream: TACTTCCAATCCATGAAAAACTGGGTTAAAG Forward: Mg++ 0 Mg++ 1.5 Mg++ 2 Mg++ 4 Mg++ 6
 * **LENGTH:** || 31 ||
 * **GC CONTENT:** || 35.5 % ||
 * **MELT TEMP:** || 57.9 ºC ||
 * **MELT TEMP:** || 65.7 ºC ||
 * **MELT TEMP:** || 65.7 ºC ||
 * **MELT TEMP:** || 66.3 ºC ||
 * **MELT TEMP:** || 66.3 ºC ||
 * **MELT TEMP:** || 67.4 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">67.4 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">68.0 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">68.0 ºC ||

<span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Downstream: TATCCACCTTTACTG TTAGTACAGGTATGCTTTT Reverse Complement of the reverse primer: AAAAGCATACCTGTACTAACAGTAAAGGTGGATA <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 0 <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 1.5 <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 2 <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 4 <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 6
 * **LENGTH:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">34 ||
 * **GC CONTENT:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">35.3 % ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">59.2 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">67.1 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">67.7 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">68.7 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">69.3 ºC ||

FASTA: TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTT

TAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACC

TGTACTTCCAAT**<span style="color: #222222; font-family: Arial,sans-serif;">CC __<span style="color: #222222; font-family: Arial,sans-serif;">ATG __**** AAAAACTGGGTTAAAGTTACCGGTGCGGGCGTTCTCTCTGCTACTCTGCTGCTCGGT ** CGG ATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCC
 * GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCAAACGTCAAGACCGAACAGACCCTG **
 * AAGCCGGGTAGCCAGATCAAACTGGAAGGTGCCGTTAATGTTCGCGACCTGGGTGGTTAC **
 * AAGACTACCGATGGTCTGACCATCAAACCGCACAAACTGATCCGCTCTGCGGAACTCGCA **
 * AACCTCTCTGACTCTGACAAAAAGAAGCTGGTTAACACCTACGACCTGTCTCACATCGTT **
 * GACTTCCGCACGTCTTCTGAAGTTGCGACCAAACCGGACCCGAAACTGACCGACGTGGAC **
 * TACACCCACGACTCTGTTATGAAAGACAACGGTACGTCTACCTCTACCCAGGACCTGACG **
 * GCGTCTCTGGCCAAGATGGACAACCCGGAAACCTTCCTGATTAACGCGAATAAATCCTTC **
 * ATTACCGACGAGACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTGGCGAAC **
 * CAGGACGGTTCCGTTCTGTGGCACTGCACCGCCGGTAAAGACCGTGCCGGTTTCGGCACC **
 * GCGCTGGTTCTGTCTGCGCTGGGTGTTGATAAAAACACCGTTATCGACGACTACATGCTG **
 * TCTAACAAATACCGTGCTGACGAAAATAAGAAAGCGATCGAAGCGGTAGCGGCGAAAACC **
 * GACAAC __ AAAAAA __ GTGATCGACGGTATGACGGCAGTTATGGAAGTTCGTGAATCTTACATC **
 * AACGCGGCGTTCGATGAGATCAATGCGAAATATGGTTCTATGGATAACTTCCTCAAAGAA **
 * AAGCTGGGCCTCACTGATGCGAAGAAAGAGCAGCTGAAA ****<span style="color: #222222; font-family: Arial,sans-serif;">ATAGGTGGAAATGAC ** AAA GCA TAC CTG TAC __TAA__ CAG TAA AGG TGG ATA

GGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCC

CTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATG

GGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTT

GCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC

GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAA

ACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTG

GAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATT

CTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATT

TAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGA

ACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAAC

TCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCC

GTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGC

GATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAG

AAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTT

CAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTG

CGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGG

CGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATG

CTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGT

CGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTA

CCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTG

ATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGG

CCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGAC

AGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA

AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC

CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG

CAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA

GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTC

TTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTG

CACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGC

GCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA

CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGA

GCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA

CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA

ACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT

GAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGC

ATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCG

CTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTC

TGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACC

GTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATG

TCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGC

GGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCAT

GGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGG

TTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGG

GTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCA

GATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAG

ACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCG

GTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGAT

CATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGA

CCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCG

CGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCAT

GATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGG

TTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGT

TGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGC

GGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTG

ATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGA

AAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTA

CCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATC

GTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGAC

ATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGC

CAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAA

TGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTC

TGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGT

CATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACA

GGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTA

ATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACT

GTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTT

TTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCG

GCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGC

GCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTAT

GCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGG

TGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAA

GCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAA

CCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCG

AAAT = =

PCR 1 was completed. The purpose of the PCR was to amplify the purple protein sequence in pGBR-22 plasmid by using M13 Reverse and Forward primers.



In lane 5 however, which was the control, there is a band, which represents contamination of DNA, or DNA spilling over from one of the wells during the electrophoresis.

=Week 4:= Shane - good on your purified DNA. - Dr. B On Tuesday, September 18th, we completed filtering out the DNA from our bacteria. This completed the collection of out pNIC-Bsa4. We then checked the concentration of the plasmid using a Nanodrop procedure.



=Week 3:= -- Good gel analysis - move your legend for the gel to underneath the image. Also mention the other procedures ( Transformation, etc.) -- Dr. B 091812

This week, I worked on a restriction enzyme digest. The enzymes involved were EcoR1 and Pvu-II. We used the NEB Buffer-2 (New England Bio Labs). The first lane was skipped, the second a ladder, third the uncut plasmid which was pgbr-22, the fourth was pgbr-22 cut by Eco-R1, the fifth was pgbr-22 cut by Pvu-II, and the sixth was pgbr-22 cut by both enzymes.

Figure 1: Due to the larger bands travelling slowly, the more fluorescent molecules show up much brighter. The fourth lane was only supposed to have one cut, the fifth was supposed to have two, and the sixth was supposed to have three. However, it seems that the Pvu-II didn't completely cut the plasmid due to there being three portions shown.

We worked on a nanodrop procedure after the electrophoresis. We then began our PCR#1.

=Week 2:=

This week we diluted primers.

We also determined which gene was present in pNIC Bsa4.

pLIC-forward: NNNNNNNNNNNNNNNNACTTTAGANGGAGANNTACATATGCACCATCATCATCATCATTCTTCTGGTGT

AGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGGAGACCGACGTCCACATATACCTGCCGTTCACTAT

TATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAAC

AGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGC

AAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAAT

AGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAG

GGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCC

ATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGA

ACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTC

AAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCAT

GATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATAAAGTTCCTGAGTTCGATTCGTCCACAAT

TAAAAATATCTCTTCTGCAAAAGGNCTGGACGTTTGGGACAGCTGGCCATTACNAAACACTGACGGCACTG

TCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGA

TTTACNTGTTCTATCAAAAAGTCNGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAA

GACAGCGANAAATTCGATGCNAATGANTCTATCCTAAANNANNNAANNNNAGAATGGNCNGGTTCANCN

NCATTTACNTCTGANGNAAAATCCNTTNNTNCNNNNNCTGATTNNNNNCNGNNANNNNTACNGCAANN

NANNNCTGANNANNNCNNNNNNNNNATCNNNNNNNNANNGNNNNTNNANNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNTNNNNNNNNNNAANNNNNNNNNANNNNNNNNNNNTNNNNNNNNNNNNAANN

GNNNNNNNNNNNN

pLIC-reverse NNNNNNNNNNNNNNNNNNNNTNNGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACG

GAGCTCGAATTCGGATCCGTATCCACCTTTACTGGAGACCGTCAATGCCAATAGGATATCGGCATTTTCTTT

TGCGTTTTTATTTGTTAACTGTTAATTGTCCTTGTTCAAGGATGCTGTCTTTGACAACAGATGTTTTCTTGC

CTTTGATGTTCAGCAGGAAGCTAGGCGCAAACGTTGATTGTTTGTCTGCGTAGAATCCTCTGTTTGTCATAT

AGCTTGTAATCACGACATTGTTTCCTTTCGCTTGAGGTACAGCGAAGTGTGAGTAAGTAAAGGTTACATCG

TTAGGATCAAGATCCATTTTTAACACAAGGCCAGTTTTGTTCAGCGGCTTGTATGGGCCAGTTAAAGAATT

AGAAACATAACCAAGCATGTAAATATCGTTAGACGTAATGCCGTCAATCGTCATTTTTGATCCGCGGGAGTC

AGTGAACAGGTACCATTTGCCGTTCATTTTAAAGACGTTCGCGCGTTCAATTTCATCTGTTACTGTGTTAGA

TGCAATCAGCGGTTTCATCACTTTTTTCAGTGTGTAATCATCGTTTAGCTCAATCATACCGAGAGCGCCGTT

TGCTAACTCAGCCGTGCGTTTTTTATCGCTTTGCAGAAGTTTTTGACTTTCTTGACGGAAGAATGATGTGCT

TTTGCCATAGTATGCTTTGTTAAATAAAGATTCTTCGCCTTGGTAGCCATCTTCAGTTCCAGTGTTTGCTTC

AAATACTAAGTATTTGTGGCCTTTATCTTCTACGTAGTGAGGATCTCTCAGCGTATGGTTGTCGCCTGAGCT

GTAGTTGCCTTCATCGATGAACTGCTGTACATTTTGATACNTTTTTCCGTCACCGTCAAAGATTGATTTATA

ATCCTCTACACCGTTGATGTTCAAAGAGCTGTCTGATGCTGATACGTTAACTTGTGCAGTTGTCAGTGTTTG

TTTGCCGTAANGNTTACCGNANAAATCANNGNANAATNAACNNATTTTTNNTCNGANGTAANNNNNGNT

GANNNGANNNTNCNTGNGNTTNGNCTTTNANNANNNNNCNTTNGCATCNNNTNNNCGCNNNNNTNNN

NANNNNNNNNNNTNNNGCNNNCNNNNNNNNNNCNANNTTNNNNNNNNNNNNNNNCNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCNNN

We also grew a DH5a strain of bacteria on SOC media.

In order to determine the DNA sequence of a plasmid, we made several comparisons. We then were able to determine the entire sequence for pGBR22.

=**Week 1:**=


 * Leishmania major **


 * *Target (protein/gene name): **** deoxyuridine triphosphatase **


 * NCBI Gene # or RefSeq#: ** **13391737**

**REFSEQ**# : 157864228


 * *Protein ID (NP or XP #) or Wolbachia#: **** 29483 **


 * *Organism: **** Leishmania major **


 * *Background/Disease Information: ** The parasite is an intracellular pathogen of the immune system targeting macrophages and dendritic cells. The disease Leishmaniasis affects the populations of 88 counties worldwide with symptoms ranging from disfiguring cutaneous and muco-cutaneous lesions that can cause widespread destruction of mucous membranes to visceral disease affecting the haemopoetic organs.


 * Essentiality of this protein: **** Gene/Ortholog: ** Tb927.7.5160 (OG4_25847); ** Phenotype: ** significant loss of fitness in differentiation of procyclic to bloodstream forms; ** Source study: ** alsford

<span style="background-color: #ffffff; font-family: arial,helvetica,clean,sans-serif;">The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric β-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding.

removes dUTP from the nucleotide triphosphate pool and therefore prevents the incorporation of uracil into the DNA, essential for replication and cell survival


 * *EC#: ** 3.6.1.23

**Link to BRENDA EC# page: []**

Figure 1: Reaction by dUTP diphosphatase


 * Enzyme Assay information **

[] ITC assay - don't have in VDS lab. Or pyrophosphate - less easy vs. phosphate. --Dr. B
 * -- link to paper that contains assay information **


 * -- List cost and quantity of substrate reagents and supplier **
 * GenScript: $282.45 Full Length Sequence **


 * -- PDB # or closest PDB entry if using homology model: 2YAY **


 * Current Inhibitors: Calcium and DUP **


 * Image of protein (PyMol or etc): **

Figure 2: Image of dUTP from PyMOL.


 * *Amino Acid Sequence: **

MKRARSANIP GAILHSLAEL QDGLNAMIDP SWRAVRSLDN WALAITMEST ELLDSYPWKW

WKNLNATPDL ANVRIELVDI FHFSLSGAMQ MRSTPDDEIP AASLKPLKEV MTTFLPAKEC

TSDPYGFVFF PLTDTQNAIA SFRNIIQLAN AYRFDVIIEC IIYAAEDLGF NLVAYYIAKH

TLNCIRQLSG YKDGSYVKVN NGVEDNSLLH NCIKDVSLDE VLDADKYVQA WNSIMANVYE

AFQIKESDRK DAERWFALAK ENRLAIKA

<span style="background-color: #ffffff; font-family: arial,helvetica,clean,sans-serif;">The enzyme has proved to be a dimer by gel filtration and is able to hydrolyse both dUTP and dUDP quite efficiently, acting as a dUTP nucleotidohydrolase (dUTPase)-dUDP nucleotidohydrolase but has a limited capacity to act upon other nucleoside di- or triphosphates.
 * Purification ** :

= ** *length of your protein in Amino Acids: 268 ** =

**Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 30353.6**


 * Molar Extinction coefficient of your protein at 280 nm wavelength: Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water. **


 * Ext. coefficient 53650 **
 * Abs 0.1% (=1 g/l) 1.768, assuming all pairs of Cys residues form cystines **

code
 * Ext. coefficient 53400 **
 * Abs 0.1% (=1 g/l) 1.759, assuming all Cys residues are reduced **

code


 * *CDS Gene Sequence **

1 ATGAAACGTGCGCGTAGCGCGAACATCCCGGGTGCCATCCTGCACTCTCTCGCGGAGCTG

61 CAGGACGGTCTCAACGCGATGATCGACCCGTCTTGGCGTGCGGTTCGTTCTCTCGACAAC

121 TGGGCCCTCGCGATCACCATGGAATCTACCGAACTGCTCGACTCCTACCCGTGGAAGTGG

181 TGGAAGAATCTGAACGCTACCCCGGACCTGGCCAATGTGCGTATCGAACTGGTTGACATC

241 TTCCATTTCTCTCTGTCTGGTGCGATGCAGATGCGTAGCACTCCGGACGATGAAATTCCA

301 GCCGCGTCCCTGAAACCGCTGAAAGAAGTTATGACCACCTTCCTGCCGGCGAAAGAATGC

361 ACCTCTGACCCGTATGGTTTCGTTTTTTTCCCGCTCACCGACACCCAGAACGCAATCGCA

421 TCTTTCCGTAATATCATCCAGCTCGCGAATGCCTACCGTTTCGACGTTATCATCGAGTGC

481 ATCATCTACGCAGCTGAAGACCTGGGTTTCAACCTGGTTGCGTACTACATCGCGAAACAC

541 ACCCTGAACTGCATCCGTCAGCTCTCTGGTTACAAAGACGGTTCTTACGTTAAAGTTAAC

601 AACGGTGTTGAAGACAACTCTCTGCTGCACAATTGCATTAAAGATGTTTCTCTGGACGAG

661 GTCCTCGACGCGGACAAATACGTTCAGGCGTGGAACTCTATCATGGCGAACGTCTACGAA

721 GCGTTCCAGATCAAAGAGTCTGACCGTAAAGACGCAGAACGTTGGTTTGCGCTGGCGAAG

781 GAAAACCGCCTGGCGATCAAGGCGTAA

Figure 3: TMpred output for dUTP indicating the protein as not a membrane bound protein.