Team:Warsaw/Calendar-Main/16 July 2009

Cloning of the mgtc promoter into the pKSII+ plasmid Kamil

Tasks:  Transformant selection 

Methods:  White colonies were picked up and transferred to a new petri dish. Liquid cultures were established along the way for plasmid purification. Both the dish and the liquid cultures were incubated at 37&deg;C overnight. 

Results:  All of the transferred colonies turned blue overnight and the purified plasmids yealded no mgtc promoter. 

Cloning of p53 coding sequence Marcin

Task:  Transformation of chemocompetent E. coli strain DH5&alpha;</li> </ul> Methods:  Ligation reaction was stopped via thermal inactivation in 80&deg;C for 20 minutes</li> Detailed protocol of transformation is described <a href="http://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>. The only modification is usage of half volume of ligation mixture prepared 15.07.09</li> </ul>

Assembly of endosomal detection operon Marcin

Task:  Restriction digest of biobricks</li> </ul> Comment: Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of biobricks were digested: <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> <a href="http://partsregistry.org/Part:BBa_C0040"> BBa_c0040</a> <a href="http://partsregistry.org/Part:BBa_C0051"> BBa_C0051</a> <a href="http://partsregistry.org/Part:BBa_E0032"> BBa_E0032</a> Methods:  Digest of BBa_B0032 using SpeI and PstI</li> Reaction mixture composition: 10 &mu;l purified plasmid DNA product 0.5 &mu;l SpeI (Fermentas) 1 &mu;l PstI (Fermentas) 5 &mu;l Buffer Tango (Fermentas) 34 &mu;l MQ water </li></ul> Digest of other biobricks using PstI and XbaI</li> Reaction mixture composition: 10 &mu;l purified plasmid DNA product 1 &mu;l XbaI (Fermentas) 1 &mu;l PstI (Fermentas) 5 &mu;l Buffer Tango (Fermentas) 34 &mu;l MQ water </li></ul> Both reaction were performed overnight (~12 hours) and both of them were subsequently inactivated via heating in 80&deg;C for 20 minutes</li> </ul></li>

Construction of K177012 operon1_part2

Ania

Tasks:


 * Set up a liquid culture from transformants containing BBa_B0032 - RBS.3 and BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid.


 * Alkaline lysis of bacterial cultures to obtain plasmid.


 * Digest of the ligated BBa_B0032 - RBS.3 and BBa_C0012 - lacI repressor  on the pSB1A2 ampicillin resistant plasmid with EcoRI and PstI to confirm ligation.

Testing different E. coli strains regarding lacI and AraC repressors
Franek

Tasks:


 * Alkaline lysis of bacterial cultures to obtain BBa_K177024 and BBa_K177025 devices


 * Transformation of Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a competent cells with BBa_K177024 and BBa_K177025

Methods:


 * 2 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing BBa_K177024 and BBa_K177025 on pSB1A2 plasmid. The cultures were incubated overnight at 37°C. Alkaline lysis was performed on both cultures, according to our standard procedure. The pellet from 5 ml of bacteria was used.


 * Chemocompetent E. coli cells from Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a strains (prepered earlier by Kuba) were transformed according to our standard procedure with either BBa_K177024 or BBa_K177025

Results:


 * Will be determined by cell colonies presence on plates