Difference between revisions of "Team:TU Darmstadt/Lab"
MaikSchork (Talk | contribs) |
MaikSchork (Talk | contribs) |
||
| Line 29: | Line 29: | ||
<center> | <center> | ||
<div class="bild" style="width:80%;"> | <div class="bild" style="width:80%;"> | ||
| + | |||
| + | |||
<svg | <svg | ||
xmlns:dc="http://purl.org/dc/elements/1.1/" | xmlns:dc="http://purl.org/dc/elements/1.1/" | ||
| Line 58: | Line 60: | ||
inkscape:current-layer="g4184" | inkscape:current-layer="g4184" | ||
inkscape:document-units="px" | inkscape:document-units="px" | ||
| − | inkscape:cy=" | + | inkscape:cy="1410.9452" |
| − | inkscape:cx=" | + | inkscape:cx="978.04393" |
| − | inkscape:zoom=" | + | inkscape:zoom="1" |
inkscape:pageshadow="2" | inkscape:pageshadow="2" | ||
inkscape:pageopacity="0" | inkscape:pageopacity="0" | ||
| Line 638: | Line 640: | ||
id="rect4328" /> | id="rect4328" /> | ||
<path | <path | ||
| − | style="display:inline;fill:#333333;fill-opacity:1;fill-rule:evenodd;stroke:none;stroke-width:3.63966417;stroke-linecap:butt;stroke-linejoin:round;stroke-miterlimit:4;stroke-dasharray:none;stroke-opacity:1" | + | style="display:inline;fill:#333333;fill-opacity:1;fill-rule:evenodd;stroke:none;stroke-width:3.63966417;stroke-linecap:butt;stroke-linejoin:round;stroke-miterlimit:4;stroke-dasharray:none;stroke-opacity:1;opacity:1" |
d="M 984.64453 141.05078 L 950.0957 203.4375 L 879.98633 163.0625 L 240.14062 531.72656 L 240.14844 602.56055 L 208.47461 602.56055 C 206.98971 602.56055 205.79492 603.7539 205.79492 605.23633 L 205.79492 612.88477 C 205.79492 614.36719 206.98971 615.56055 208.47461 615.56055 L 240.15039 615.56055 L 240.18555 941.47656 L 178.04883 941.17969 L 178.25 898.66602 L 189.38672 898.76562 C 189.98497 897.9013 189.99642 896.49791 189.4082 895.62109 L 171.98633 869.65234 C 171.39834 868.77559 170.44096 868.76465 169.83984 869.62891 L 151.99805 895.28711 C 151.69905 895.71932 151.5487 896.28539 151.54492 896.85352 C 151.54499 897.42152 151.68434 897.99319 151.97852 898.43164 L 164.25195 898.53906 L 164.04883 941.11328 L 163.9668 958.11328 L 240.1875 958.47656 L 240.21094 1168.5859 L 176.69727 1168.3105 L 176.88086 1125.7969 L 188.01758 1125.8906 C 188.61544 1125.026 188.62566 1123.6227 188.03711 1122.7461 L 170.60352 1096.7871 C 170.01514 1095.9106 169.05776 1095.8991 168.45703 1096.7637 L 150.62891 1122.4316 C 150.3301 1122.864 150.17736 1123.4299 150.17383 1123.998 C 150.17443 1124.5661 150.31497 1125.1359 150.60938 1125.5742 L 162.88086 1125.6777 L 162.69727 1168.25 L 162.62305 1185.25 L 240.21289 1185.5859 L 240.22266 1269.8281 L 436.84766 1383.5098 L 421.61914 1409.877 C 420.87836 1411.1596 421.3159 1412.7879 422.59961 1413.5293 L 429.22266 1417.3555 C 430.50637 1418.0969 432.13618 1417.6596 432.87695 1416.377 L 448.10156 1390.0156 L 880.0332 1639.7441 L 969.51562 1588.1914 L 986.00195 1616.7363 C 986.74273 1618.019 988.37254 1618.4543 989.65625 1617.7129 L 996.2793 1613.8887 C 997.56301 1613.1473 998.00054 1611.517 997.25977 1610.2344 L 980.78125 1581.7012 L 1433.2676 1321.0137 L 1465.0469 1375.7109 L 1428.2852 1397.0684 L 1422.7305 1387.416 C 1421.6822 1387.3379 1420.4659 1388.039 1420.0078 1388.9902 L 1406.4414 1417.1641 C 1405.9832 1418.1151 1406.4582 1418.9468 1407.5078 1419.0273 L 1438.668 1421.416 C 1439.1921 1421.4549 1439.7582 1421.2982 1440.25 1421.0137 C 1440.7397 1420.7259 1441.1596 1420.3175 1441.3887 1419.8418 L 1435.2676 1409.2031 L 1472.0801 1387.8164 L 1486.7773 1379.2754 L 1447.998 1312.5273 L 1520.2891 1270.8789 L 1520.4023 1175.0254 L 1552.1914 1175.0254 C 1553.6763 1175.0254 1554.8711 1173.832 1554.8711 1172.3496 L 1554.8711 1164.7031 C 1554.8711 1163.2207 1553.6763 1162.0273 1552.1914 1162.0273 L 1520.418 1162.0273 L 1520.7129 915.18555 L 1585.2754 915.62109 L 1584.9883 958.13477 L 1573.8535 958.01172 C 1573.2536 958.87482 1573.2397 960.27827 1573.8262 961.15625 L 1591.1953 987.16016 C 1591.7816 988.0381 1592.739 988.05029 1593.3418 987.1875 L 1611.2344 961.56445 C 1611.5344 961.1329 1611.6865 960.56618 1611.6914 959.99805 C 1611.6924 959.43003 1611.555 958.86086 1611.2617 958.42188 L 1598.9883 958.28711 L 1599.2754 915.71484 L 1599.3906 898.71484 L 1520.7324 898.18359 L 1520.7656 871.02734 L 1552.1914 871.02734 C 1553.6763 871.02734 1554.8711 869.83399 1554.8711 868.35156 L 1554.8711 860.70312 C 1554.8711 859.2207 1553.6763 858.02734 1552.1914 858.02734 L 1520.7812 858.02734 L 1521.0781 609.1875 L 1585.2754 609.62109 L 1584.9883 652.13477 L 1573.8535 652.01172 C 1573.2536 652.87482 1573.2397 654.27827 1573.8262 655.15625 L 1591.1953 681.16016 C 1591.7816 682.0381 1592.739 682.05029 1593.3418 681.1875 L 1611.2344 655.56445 C 1611.5344 655.1329 1611.6865 654.56618 1611.6914 653.99805 C 1611.6924 653.43003 1611.555 652.86086 1611.2617 652.42188 L 1598.9883 652.28711 L 1599.2754 609.71484 L 1599.3906 592.71484 L 1521.0977 592.18555 L 1521.1699 532.30469 L 1433.1582 481.62109 L 1448.7461 454.63281 C 1449.4869 453.35017 1449.0493 451.71991 1447.7656 450.97852 L 1441.1426 447.1543 C 1439.8589 446.4129 1438.2291 446.84822 1437.4883 448.13086 L 1421.8926 475.13281 L 1242.5723 371.86719 L 1270.5254 321.39258 L 1307.7188 341.99023 L 1302.2812 351.70898 C 1302.752 352.64886 1303.9778 353.33298 1305.0293 353.23828 L 1336.1738 350.42969 C 1337.2253 350.33504 1337.6958 349.50074 1337.2266 348.55859 L 1323.2891 320.58594 C 1323.0537 320.116 1322.6293 319.71174 1322.1328 319.43555 C 1321.6365 319.1594 1321.0687 319.01123 1320.543 319.05859 L 1314.5527 329.77148 L 1277.3086 309.14453 L 1262.4355 300.9082 L 1227.8379 363.38281 L 964.82812 211.92188 L 992.73242 161.53516 L 1029.9258 182.13281 L 1024.4902 191.85156 C 1024.961 192.79146 1026.1868 193.47557 1027.2383 193.38086 L 1058.3809 190.57227 C 1059.4323 190.47766 1059.9028 189.64337 1059.4336 188.70117 L 1045.4961 160.72852 C 1045.2607 160.25857 1044.8383 159.85447 1044.3418 159.57812 L 1044.3418 159.57617 C 1043.8454 159.30001 1043.2758 159.15379 1042.75 159.20117 L 1036.7598 169.91211 L 999.51562 149.28711 L 984.64453 141.05078 z M 880.02344 202.64062 L 1404.75 504.81836 L 1387.248 535.12109 C 1386.5073 536.40374 1386.9428 538.03399 1388.2266 538.77539 L 1394.8496 542.59961 C 1396.1333 543.34101 1397.7631 542.90569 1398.5039 541.62305 L 1416.0137 511.30469 L 1486.8359 552.08984 L 1486.4707 858.02734 L 1451.748 858.02734 C 1450.2631 858.02734 1449.0684 859.2207 1449.0684 860.70312 L 1449.0684 868.35156 C 1449.0684 869.83399 1450.2631 871.02734 1451.748 871.02734 L 1486.4551 871.02734 L 1486.1074 1162.0273 L 1451.748 1162.0273 C 1450.2631 1162.0273 1449.0684 1163.2207 1449.0684 1164.7031 L 1449.0684 1172.3496 C 1449.0684 1173.832 1450.2631 1175.0254 1451.748 1175.0254 L 1486.0918 1175.0254 L 1486.002 1251.0742 L 963.63867 1552.0195 L 947.01953 1523.2441 C 946.27875 1521.9615 944.64895 1521.5262 943.36523 1522.2676 L 936.74219 1526.0918 C 935.45847 1526.8332 935.02094 1528.4635 935.76172 1529.7461 L 952.37305 1558.5078 L 880.06641 1600.166 L 465.24609 1360.332 L 483.11719 1329.3867 C 483.85797 1328.1041 483.42043 1326.4758 482.13672 1325.7344 L 475.51367 1321.9082 C 474.22996 1321.1668 472.60015 1321.6021 471.85938 1322.8848 L 453.99023 1353.8242 L 274.55273 1250.0801 L 274.48242 615.56055 L 308.91797 615.56055 C 310.40287 615.56055 311.59766 614.36719 311.59766 612.88477 L 311.59766 605.23633 C 311.59766 603.7539 310.40287 602.56055 308.91797 602.56055 L 274.48047 602.56055 L 274.47461 551.54297 L 880.02344 202.64062 z M 1384.5 667.1543 L 1384.5 674.1543 L 1384.5 912.71289 L 1391.5 912.71289 L 1436.7617 912.71289 L 1436.7617 930.85352 C 1436.7617 933.00447 1438.4958 934.73633 1440.6504 934.73633 L 1445.8828 934.73633 C 1448.0374 934.73633 1449.7715 933.00447 1449.7715 930.85352 L 1449.7715 887.57422 C 1449.7715 885.42326 1448.0374 883.69141 1445.8828 883.69141 L 1440.6504 883.69141 C 1438.4958 883.69141 1436.7617 885.42326 1436.7617 887.57422 L 1436.7617 905.71289 L 1391.5 905.71289 L 1391.5 674.1543 L 1448.5 674.1543 L 1448.5 667.1543 L 1384.5 667.1543 z " | d="M 984.64453 141.05078 L 950.0957 203.4375 L 879.98633 163.0625 L 240.14062 531.72656 L 240.14844 602.56055 L 208.47461 602.56055 C 206.98971 602.56055 205.79492 603.7539 205.79492 605.23633 L 205.79492 612.88477 C 205.79492 614.36719 206.98971 615.56055 208.47461 615.56055 L 240.15039 615.56055 L 240.18555 941.47656 L 178.04883 941.17969 L 178.25 898.66602 L 189.38672 898.76562 C 189.98497 897.9013 189.99642 896.49791 189.4082 895.62109 L 171.98633 869.65234 C 171.39834 868.77559 170.44096 868.76465 169.83984 869.62891 L 151.99805 895.28711 C 151.69905 895.71932 151.5487 896.28539 151.54492 896.85352 C 151.54499 897.42152 151.68434 897.99319 151.97852 898.43164 L 164.25195 898.53906 L 164.04883 941.11328 L 163.9668 958.11328 L 240.1875 958.47656 L 240.21094 1168.5859 L 176.69727 1168.3105 L 176.88086 1125.7969 L 188.01758 1125.8906 C 188.61544 1125.026 188.62566 1123.6227 188.03711 1122.7461 L 170.60352 1096.7871 C 170.01514 1095.9106 169.05776 1095.8991 168.45703 1096.7637 L 150.62891 1122.4316 C 150.3301 1122.864 150.17736 1123.4299 150.17383 1123.998 C 150.17443 1124.5661 150.31497 1125.1359 150.60938 1125.5742 L 162.88086 1125.6777 L 162.69727 1168.25 L 162.62305 1185.25 L 240.21289 1185.5859 L 240.22266 1269.8281 L 436.84766 1383.5098 L 421.61914 1409.877 C 420.87836 1411.1596 421.3159 1412.7879 422.59961 1413.5293 L 429.22266 1417.3555 C 430.50637 1418.0969 432.13618 1417.6596 432.87695 1416.377 L 448.10156 1390.0156 L 880.0332 1639.7441 L 969.51562 1588.1914 L 986.00195 1616.7363 C 986.74273 1618.019 988.37254 1618.4543 989.65625 1617.7129 L 996.2793 1613.8887 C 997.56301 1613.1473 998.00054 1611.517 997.25977 1610.2344 L 980.78125 1581.7012 L 1433.2676 1321.0137 L 1465.0469 1375.7109 L 1428.2852 1397.0684 L 1422.7305 1387.416 C 1421.6822 1387.3379 1420.4659 1388.039 1420.0078 1388.9902 L 1406.4414 1417.1641 C 1405.9832 1418.1151 1406.4582 1418.9468 1407.5078 1419.0273 L 1438.668 1421.416 C 1439.1921 1421.4549 1439.7582 1421.2982 1440.25 1421.0137 C 1440.7397 1420.7259 1441.1596 1420.3175 1441.3887 1419.8418 L 1435.2676 1409.2031 L 1472.0801 1387.8164 L 1486.7773 1379.2754 L 1447.998 1312.5273 L 1520.2891 1270.8789 L 1520.4023 1175.0254 L 1552.1914 1175.0254 C 1553.6763 1175.0254 1554.8711 1173.832 1554.8711 1172.3496 L 1554.8711 1164.7031 C 1554.8711 1163.2207 1553.6763 1162.0273 1552.1914 1162.0273 L 1520.418 1162.0273 L 1520.7129 915.18555 L 1585.2754 915.62109 L 1584.9883 958.13477 L 1573.8535 958.01172 C 1573.2536 958.87482 1573.2397 960.27827 1573.8262 961.15625 L 1591.1953 987.16016 C 1591.7816 988.0381 1592.739 988.05029 1593.3418 987.1875 L 1611.2344 961.56445 C 1611.5344 961.1329 1611.6865 960.56618 1611.6914 959.99805 C 1611.6924 959.43003 1611.555 958.86086 1611.2617 958.42188 L 1598.9883 958.28711 L 1599.2754 915.71484 L 1599.3906 898.71484 L 1520.7324 898.18359 L 1520.7656 871.02734 L 1552.1914 871.02734 C 1553.6763 871.02734 1554.8711 869.83399 1554.8711 868.35156 L 1554.8711 860.70312 C 1554.8711 859.2207 1553.6763 858.02734 1552.1914 858.02734 L 1520.7812 858.02734 L 1521.0781 609.1875 L 1585.2754 609.62109 L 1584.9883 652.13477 L 1573.8535 652.01172 C 1573.2536 652.87482 1573.2397 654.27827 1573.8262 655.15625 L 1591.1953 681.16016 C 1591.7816 682.0381 1592.739 682.05029 1593.3418 681.1875 L 1611.2344 655.56445 C 1611.5344 655.1329 1611.6865 654.56618 1611.6914 653.99805 C 1611.6924 653.43003 1611.555 652.86086 1611.2617 652.42188 L 1598.9883 652.28711 L 1599.2754 609.71484 L 1599.3906 592.71484 L 1521.0977 592.18555 L 1521.1699 532.30469 L 1433.1582 481.62109 L 1448.7461 454.63281 C 1449.4869 453.35017 1449.0493 451.71991 1447.7656 450.97852 L 1441.1426 447.1543 C 1439.8589 446.4129 1438.2291 446.84822 1437.4883 448.13086 L 1421.8926 475.13281 L 1242.5723 371.86719 L 1270.5254 321.39258 L 1307.7188 341.99023 L 1302.2812 351.70898 C 1302.752 352.64886 1303.9778 353.33298 1305.0293 353.23828 L 1336.1738 350.42969 C 1337.2253 350.33504 1337.6958 349.50074 1337.2266 348.55859 L 1323.2891 320.58594 C 1323.0537 320.116 1322.6293 319.71174 1322.1328 319.43555 C 1321.6365 319.1594 1321.0687 319.01123 1320.543 319.05859 L 1314.5527 329.77148 L 1277.3086 309.14453 L 1262.4355 300.9082 L 1227.8379 363.38281 L 964.82812 211.92188 L 992.73242 161.53516 L 1029.9258 182.13281 L 1024.4902 191.85156 C 1024.961 192.79146 1026.1868 193.47557 1027.2383 193.38086 L 1058.3809 190.57227 C 1059.4323 190.47766 1059.9028 189.64337 1059.4336 188.70117 L 1045.4961 160.72852 C 1045.2607 160.25857 1044.8383 159.85447 1044.3418 159.57812 L 1044.3418 159.57617 C 1043.8454 159.30001 1043.2758 159.15379 1042.75 159.20117 L 1036.7598 169.91211 L 999.51562 149.28711 L 984.64453 141.05078 z M 880.02344 202.64062 L 1404.75 504.81836 L 1387.248 535.12109 C 1386.5073 536.40374 1386.9428 538.03399 1388.2266 538.77539 L 1394.8496 542.59961 C 1396.1333 543.34101 1397.7631 542.90569 1398.5039 541.62305 L 1416.0137 511.30469 L 1486.8359 552.08984 L 1486.4707 858.02734 L 1451.748 858.02734 C 1450.2631 858.02734 1449.0684 859.2207 1449.0684 860.70312 L 1449.0684 868.35156 C 1449.0684 869.83399 1450.2631 871.02734 1451.748 871.02734 L 1486.4551 871.02734 L 1486.1074 1162.0273 L 1451.748 1162.0273 C 1450.2631 1162.0273 1449.0684 1163.2207 1449.0684 1164.7031 L 1449.0684 1172.3496 C 1449.0684 1173.832 1450.2631 1175.0254 1451.748 1175.0254 L 1486.0918 1175.0254 L 1486.002 1251.0742 L 963.63867 1552.0195 L 947.01953 1523.2441 C 946.27875 1521.9615 944.64895 1521.5262 943.36523 1522.2676 L 936.74219 1526.0918 C 935.45847 1526.8332 935.02094 1528.4635 935.76172 1529.7461 L 952.37305 1558.5078 L 880.06641 1600.166 L 465.24609 1360.332 L 483.11719 1329.3867 C 483.85797 1328.1041 483.42043 1326.4758 482.13672 1325.7344 L 475.51367 1321.9082 C 474.22996 1321.1668 472.60015 1321.6021 471.85938 1322.8848 L 453.99023 1353.8242 L 274.55273 1250.0801 L 274.48242 615.56055 L 308.91797 615.56055 C 310.40287 615.56055 311.59766 614.36719 311.59766 612.88477 L 311.59766 605.23633 C 311.59766 603.7539 310.40287 602.56055 308.91797 602.56055 L 274.48047 602.56055 L 274.47461 551.54297 L 880.02344 202.64062 z M 1384.5 667.1543 L 1384.5 674.1543 L 1384.5 912.71289 L 1391.5 912.71289 L 1436.7617 912.71289 L 1436.7617 930.85352 C 1436.7617 933.00447 1438.4958 934.73633 1440.6504 934.73633 L 1445.8828 934.73633 C 1448.0374 934.73633 1449.7715 933.00447 1449.7715 930.85352 L 1449.7715 887.57422 C 1449.7715 885.42326 1448.0374 883.69141 1445.8828 883.69141 L 1440.6504 883.69141 C 1438.4958 883.69141 1436.7617 885.42326 1436.7617 887.57422 L 1436.7617 905.71289 L 1391.5 905.71289 L 1391.5 674.1543 L 1448.5 674.1543 L 1448.5 667.1543 L 1384.5 667.1543 z " | ||
transform="matrix(1.8661755,-0.00328477,0.00329025,1.8692949,-544.79679,-1548.1924)" | transform="matrix(1.8661755,-0.00328477,0.00329025,1.8692949,-544.79679,-1548.1924)" | ||
| Line 647: | Line 649: | ||
transform="matrix(1.8661755,-0.00328477,0.00329025,1.8692949,-544.79679,-1548.1924)" | transform="matrix(1.8661755,-0.00328477,0.00329025,1.8692949,-544.79679,-1548.1924)" | ||
id="rect4341" /> | id="rect4341" /> | ||
| + | <path | ||
| + | style="opacity:1;fill:#ffffff;fill-opacity:1;stroke:none;stroke-width:2;stroke-linecap:round;stroke-linejoin:miter;stroke-miterlimit:4;stroke-dasharray:none;stroke-dashoffset:0;stroke-opacity:1" | ||
| + | d="M 994.19141 216.14844 C 990.05985 216.02809 986.00099 218.12144 983.76758 221.96484 L 963.95898 256.05273 C 960.71042 261.6432 962.58887 268.75488 968.16992 271.99805 L 1127.1055 364.35547 C 1128.6591 365.25829 1130.3298 365.74853 1132.0039 365.88672 L 1123.6074 380.27734 C 1124.5097 382.55902 1127.1988 384.12412 1129.6348 383.78516 L 1201.791 373.74414 C 1204.228 373.4057 1205.4631 371.29713 1204.5605 369.01562 L 1177.7715 301.28516 C 1176.8692 299.00348 1174.182 297.43845 1171.7461 297.77734 L 1162.4648 313.68359 C 1161.5088 312.25522 1160.2316 311.01046 1158.6406 310.08594 L 999.70508 217.72852 C 997.96099 216.71502 996.06943 216.20309 994.19141 216.14844 z " | ||
| + | transform="matrix(1.8661755,-0.00328477,0.00329025,1.8692949,-544.79679,-1548.1924)" | ||
| + | id="rect4306" /> | ||
<g | <g | ||
id="aaRS" | id="aaRS" | ||
| − | inkscape:label="# | + | inkscape:label="#g4309"> |
| − | < | + | <path |
| − | + | inkscape:connector-curvature="0" | |
| − | + | id="rect4300" | |
| − | + | d="m 1311.25,-1147.4129 c -7.7106,-0.2114 -15.2782,3.715 -19.4335,10.9068 l -36.8542,63.7853 c -6.044,10.4609 -2.5151,23.7486 7.9108,29.7927 l 296.9056,172.12121 c 2.9023,1.68253 6.0217,2.59344 9.1463,2.84626 l -15.622,26.9279 c 1.6914,4.26217 6.7149,7.17898 11.2597,6.53735 l 134.6231,-19.00665 c 4.5468,-0.64065 6.8446,-4.58624 5.1528,-8.84809 l -50.2158,-126.52021 c -1.6913,-4.26217 -6.7113,-7.17886 -11.256,-6.53736 l -17.2682,29.76396 c -1.7889,-2.66692 -4.1763,-4.98953 -7.1484,-6.71251 l -296.9055,-172.12116 c -3.2581,-1.8888 -6.7898,-2.8395 -10.2947,-2.9355 z" | |
| − | + | style="opacity:1;fill:#00a86b;fill-opacity:1;fill-rule:nonzero;stroke:none;stroke-width:7;stroke-linecap:round;stroke-linejoin:round;stroke-miterlimit:4;stroke-dasharray:none;stroke-opacity:1" /> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
<text | <text | ||
transform="matrix(0.86093697,0.50792138,-0.50706877,0.86237316,0,0)" | transform="matrix(0.86093697,0.50792138,-0.50706877,0.86237316,0,0)" | ||
Latest revision as of 01:43, 20 October 2016
WET LAB
THE PROJECT
The Colicide Squad is a biosafety system which makes bacterial strains survive only in medium containing a specific non-natural amino acid (nnAA). The genetic circuit requires four parts:
- Orthogonal Pair for the incorporation of the nnAA
- mVenus as a reporter for low levels of this nnAA
- Colicin E2 as a DNA degrading toxin, activated by the absence of the nnAA
- λ-Phage Integration for genomic integration of GFP as readout for metabolic burden
Figure 1: The entire genetic circuit of this year's project
INCORPORATION OF A NON-NATURAL AMINO ACID
ABSTRACT
In order to detect the presence of a specific non-natural amino acid (nnAA) in vivo the concept of amber suppression is used. This means the occurrence of the amber stop codon (UAG) in an open reading frame does not cancel protein translation but codes for a specific nnAA, in our case O-methyl-l-tyrosine (OMT). However, for the incorporation the nnAA needs to be present in the medium, otherwise the translation stops. The mechanism requires a tRNA with an anticodon complementary to the amber stop codon and an aminoacyl RNA synthetase (aaRS) loading the tRNA with the specific nnAA. The tRNA and aaRS combination is called an 'orthogonal pair'.
REPORTING LOW nnAA LEVELS
ABSTRACT
Glow before you go - What does this actually mean? The aim of our project is to make biology safer by introducing a suicide system into E. coli. Before the suicide is triggered, a reporter protein is expressed to indicate the release of E. coli or to show a deficiency of the non-natural amino acid in the surrounding medium which is necessary for the bacteria to survive. As a reporter protein, we chose mVenus which is a mutant of eYFP. mVenus is located downstream of a promoter which is repressed by a dimeric protein, the Zif23-GCN4 repressor. This repressor carries an amber mutation at position 4 (F4OMT). As a result, the non-natural amino acid O-methyl-l-tyrosine (OMT) is integrated into the protein sequence as long as there is enough OMT in the medium. With decreasing OMT concentration, the translation of the repressor stops due to the early amber stop codon and the repressor cannot bind to the promoter. This leads to the expression of the reporter protein mVenus which can be detected by fluorescence measurements.
KILL(SWITCH)
ABSTRACT
Synthetic suicide systems have been chosen safeguards in synthetic biology since the field exists. There are different kinds of designs, often based on a regulating mechanism and a host killing toxin or different kinds of inhibition of metabolic pathways. However, these mechanisms most often do not tackle the problem of synthetic DNA surviving the death of host cells. Here we show a possible design for a simple synthetic killswitch based on the endonuclease Colicin E2 and its corresponding suppressing protein Im2. Endonuclease and suppression protein expression is regulated by amber suppression. Therefore, an amber stop codon coding for the non-natural amino acid O-methyl-l-tyrosine is implemented into the gene. The aim of the system is not to simply kill the host, but also to degrade all DNA within the cell and its surroundings, preventing the escape of transgenic DNA.
METABOLIC BURDEN
ABSTRACT
Artificial plasmids are a significant burden to the host. The design of pathways, e.g. the combination of different promoter and RBS systems, results in different amounts of product. Measurement of the metabolic burden is a key for quantitative optimization of metabolic engineering approaches. We want to establish a new approach to iGEM by providing a measurement strain to the community. As described by F. Ceroni et al., we integrated one copy of the GFP gene into the genome of E. coli, which offers us a highly accurate and instantaneous measurement of the impact of our plasmids on the host. Metabolic burden measurement is of economical interest, because it enables academic and industrial research testing several different pathways at once in a short period of time by using microplate reader. For the integration, we used the λ‑Integrase site‑specific recombination pathway, described by A. Landy in 2015. Therefore, we designed two plasmids (BBa_K1976000 and BBa_K1976001) and measured them using single cell measurement via microplate reader.
CHEMICAL SYNTHESIS
ABSTRACT
Since non‑natural amino acids are expensive in comparison to natural amino acids, we searched for a high yielding synthesis of O-methyl-l-tyrosine. When chemically altering an amino acid to a non‑natural derivate the higher reactivity of the amino and carboxyl groups in comparison to the desired reactive group has to be considered. For this reason, amino and carboxyl groups need to be protected before carrying out the desired synthesis.
For the protection of the amino group an acetylation reaction was performed to form N‑acetyl‑L‑tyrosine, which was then methylated at the carboxyl group and the hydroxyl group using dimethyl sulfate in a Williamson ether synthesis. In order to finally form the non‑natural amino acid, an acidic hydrolysis using hydrochloric acid was performed.
