IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/16

iGEM Project name 1

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Misc Jason 48-hr staph competent cell transformant didn't show growth Redo using staph shuttle vector Eric F. Made 800mL LB Broth and autoclaved - probably still in the autoclave Low on glassware - waiting on auto-autoclave Low on some supplies, put on order sheet Biofilm Track Eric F. Biofilm Growth Protocol Day 3 Removed from incubator at 1200 (25 hrs of incubation) Control in F7 showed growth, surrounding control wells did not Specks visible in D8 and D10 Will continue experiment ignoring results from: D8 D10 E8 - adjacent to contaminated control F8 - adjacent to contaminated control Placed in bio safety cabinet to dry overnight @ 1440 Biofilm Growth Protocol Day 1 3 - 3mL plastic test tubes 1 control 8325-4 RN4220 Into incubator w/o shaking @ 1630 Biofilm (100811) Growth Protocol Day 5+ Took OD readings of Test 1 plate (100811a - different glucose %s) and Test 2 plate (100811b - all locations) Test 1 - 100811a - OD550 Readings 25 <> 3 4 5 6 7 8 9 10 26 C 0.1052 0.1602 0.1652 0.1402 0.1432 0.1454 0.1769 0.0945 27 D 0.1736 0.1404 0.1528 0.0964 0.1629 0.0974 0.1596 0.1941 28 E 0.2193 0.1934 0.1758 0.1013 0.1759 0.1916 0.1981 0.1949 29 F 0.0977 0.1874 0.1776 0.1997 0.1656 0.1815 0.186 0.0967 Where: Red = Control at that row's Glucose level Row C = 4% Glucose - RN4220 Row D = 2% Glucose - RN4220 Row E = 1% Glucose - RN4220 Row F = 0.5% Gluocse - RN4220 Test 2 - 100811b - OD550 Readings 24 <> 1 2 3 4 5 6 7 8 9 10 11 12 25 A 0.2131 0.1653 0.1571 0.1857 0.1535 0.1532 0.1423 0.1573 0.1678 0.2214 0.1711 0.152 26 B 0.201 0.2033 0.1522 0.151 0.1429 0.1834 0.152 0.1574 0.1609 0.1633 0.1213 0.1499 27 C 0.198 0.1578 0.1561 0.1541 0.0927 0.1646 0.1497 0.1536 0.1507 0.1609 0.1763 0.1882 28 D 0.1929 0.1983 0.1703 0.1542 0.1465 0.1528 0.1506 0.1492 0.1593 0.1723 0.1558 0.1703 29 E 0.1601 0.178 0.1611 0.1407 0.1673 0.1611 0.1516 0.1492 0.1658 0.168 0.1668 0.1932 30 F 0.2193 0.1551 0.1721 0.1531 0.1731 0.1686 0.1608 0.1613 0.1696 0.1444 0.1573 0.1673 31 G 0.1875 0.165 0.1592 0.159 0.1534 0.1365 0.1681 0.1556 0.182 0.1536 0.0898 0.1627 32 H 0.1667 0.1863 0.1668 0.1943 0.1776 0.1687 0.1867 0.1869 0.1487 0.1749 0.1692 0.1764 Where: Red = Control All rows and columns are RN4220 w/ 4% glucose Conclusions 1% has the highest thickness biofilm Edges show more biofilm growth SUCCESS! Even though we already declared failure. Quorum Sensing Track Phillip Digested and ligated agr CA (natural RBS from PCR product; cut at EP)with PSB1C3 (cut at EP) following NEB biobrick protocols. Digested and ligated agrCA natural RBS from PCR product; cut at ES) with B0014 (cut at XP) and PSB1Ce (cut at EP) following NEB biobrick protocls. Started O/N culture of Staph aureus NCTC 8325 for AIP generation. TSB, 37 °C, 180 rpm. Transformed ligation mixtures from above into E. coli competent DH5(alpha, following UBC iGEM procedures (e.g. incubate for 1 hour at 37°C, no shaking, in LB). Plated onto LB chloramphenicol agar