Team:Warsaw/Calendar-Main/17 August 2009

Assembly of endosomal detection operon Marcin Task 1:  Isolation of  pSB1A3 plasmids containing  BBa_B0032 and  BBa_E0022  Methods:  Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here Task 2:  Digest previously isolated plasmids to verify the correctness of the ligation</li></ul> Methods:   Reaction mixture composition:</li> 1 &mu;l purified plasmid DNA product 0.5 &mu;l XbaI (Fermentas) 0.5 &mu;l PstI (Fermentas) 2 &mu;l Buffer Tango (Fermentas) 16.5 &mu;l MQ water </ul> Results: <img src="http://2009.igem.org/wiki/images/3/3d/Digest_17_08_09.png" width="60%" heigth="60%"> Comment:  All isolated plasmids had the expected construct. Task 3:  Prepare bacterial cultures to isolate following constructs:</li> p53 CDS and  BBa_B0032</a> on  pSB1A3</a> plasmid</li> cro CDS and  BBa_B0032</a> on  pSB1A3</a> plasmid</li></ul></ul> Task 4:  Isolate of following constructs:</li> <li>p53 CDS and <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid</li> <li>cro CDS and <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid</li></ul></ul> Methods: <ul> <li>Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described <a href="http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf" here>here</a></li></ul> Task 5: <ul> <li>Digest previously isolated plasmids to verify the correctness of the ligation</li></ul> Methods: <ul> <li> Reaction mixture composition:</li> 1 &mu;l purified plasmid DNA product 0.5 &mu;l XbaI (Fermentas) 0.5 &mu;l PstI (Fermentas) 2 &mu;l Buffer Tango (Fermentas) 16.5 &mu;l MQ water </ul> Results: <img src="http://2009.igem.org/wiki/images/1/1e/Isolation_17_08_09.png" width="60%" heigth="60%"> Comment:  None of isolated plasmids had the expected constructs. Ligations must be prepared once again. Task 6: <ul> <li>Restriction digest of following constructs:</li> <ul><li>p53 CDS on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid using XbaI and PstI</li> <li><a href="http://partsregistry.org/Part:BBa_R0080"> BBa_R0080</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid using SpeI and PstI</li> <li><a href="http://partsregistry.org/Part:BBa_E0010"> BBa_E0010</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid using PstI and XbaI</li> <li><a href="http://partsregistry.org/Part:BBa_C0040"> BBa_C0040</a> with <a href="http://partsregistry.org/Part:BBa_E0032"> RBS</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid</li></ul></ul> Methods: <ul> <li> Reaction mixture composition:</li> 20 &mu;l purified plasmid DNA product 1 &mu;l XbaI (Fermentas) or 0.5 SpeI (Fermentas) 1 &mu;l PstI (Fermentas) 5 &mu;l Buffer Tango (Fermentas) 24 &mu;l MQ water </ul> Results: <img src="http://2009.igem.org/wiki/images/0/09/PSB_p53_R0080_C0040%2BRBS_digest_18_08_09.png" width="60%" heigth="65%">

Making of the plac-RBS-llo part Jarek

Tasks: <ul> <li>Transformating the competent E.coli strain TopF' with ligation (acquired from Marek) containing RBS-llo gene cloned into vector with plac promoter </li> <li>Plating the transformated culure on LB+Amp plates</li> </ul>

Cloning switch 1 regulatory parts [ <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012">K177012</a>-PcI.RBS.LacI, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177033">K177033</a>-PcI.RBS.LacI.PcI.RBS.RFP.terminator, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177011">K177011</a>-PLacI.RBS.cI.terminator, <a href="http://partsregistry.org/Part:BBa_K177038">K177038</a>-PLacI.RBS.cI.terminator.PLacI.RBS.GFP.terminator ] into two compatible low copy number plasmids of different antibiotic resistance Ania Tasks: <ul> <li> </li> </ul>