Team:Warsaw/Calendar-Main/17 July 2009

Assembly of endosomal detection operon Marcin Task 1:  Gel-out of digested biobricks  Methods  After the digest samples were loaded into gel and electrophoretically separated Image of the digested biobricks:  verification of the effectiveness of the restriction digest  Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described here Quantification of concentration of p53 DNA after restriction digest:  1 &mu;l of the digest mixture was diluted to 10 &mu;l and loaded into the gel. <img src="http://2009.igem.org/wiki/images/7/77/Biobricks_gel-out_evaluation_17_07_09.png"> quantification DNA abundance after gel-out Destription of the gel lane 1 - B0032 lane 2 - C0040 lane 3 - C0051 lane 4 - E0032 Task 2:  Ligations of biobricks</li> </ul> Comment: Due to obtain set of biobricks which each of them contain RBS and particular coding sequence I designed the following elemets (the order of biobricks is from the 5'end to 3' end): <ol> <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a>+<a href="http://partsregistry.org/Part:BBa_C0040"> BBa_c0040</a> </li> <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a>+<a href="http://partsregistry.org/Part:BBa_C0051"> BBa_C0051</a> </li> <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a>+<a href="http://partsregistry.org/Part:BBa_E0032"> BBa_E0032</a> </li> <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> (ligation of empty vector as a negative control of the experiment) </ol> Methods:  Ligation mixture composition: 3 &mu;l digested plasmid with RBS 3 &mu;l digested CDS 5 &mu;l ligation buffer (Fermentas, PEG4000 have been added previously) 1 &mu;l ligase T4 (Fermentas) 13 &mu;l MQ water </li> Negative control mixture composition: 3 &mu;l digested plasmid with RBS 5 &mu;l ligation buffer (Fermentas, PEG4000 have been added previously) 1 &mu;l ligase T4 (Fermentas) 16 &mu;l MQ water </li> Duration of ligation was about 8 hours</li> </ul> Task 3:  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 ligation is described <a href="http://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>. The only modification is total volume of ligation mixture prepared 16.07.09</li> petri dish were hold in 37&deg;C for 36 hours</li> </ul>

Cloning of p53 coding sequence Marcin

Task: <ul> <li>Isolation and evaluation of pKS/p53 plasmid</li> </ul>  Comment:  Besides high effectivity of transformation all of the bacterial colonies were blue. It is possible that excess of X-Gal may cause some problems with the selection system. Due to this uncentainty I decide to prepare a few bacterial breedings to isolate the plasmids and digest them. Methods: <ul> <ol> <li>Prepare LB medium with kanamycin</li> <li>Add 3.5 ml of the medium to the probes</li> <li>Add one bacterial colony to each probe</li> <li>Breed the bacteria about 8 hours</li> </ol> </ol> </ul> </li> Construction of K177012 operon1_part2 Franek Jarek Jarek and Franek run the gel Ania only described it. Thank you guys:) Tasks:


 * Samples of ligatedBBa_B0032 - RBS.3 and BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid cut with EcoRI and PstI were run on the gel.

Results: Obtain results confirm ligation of the http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and BBa_C0012 - lacI repressor, the undigested plasmid is visible on the gel. It is probably due to incorrect digestion buffer concentration.



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

Tasks:


 * Seting new liquid cultures to obtain of pure BBa_K177024 construct

Methods:


 * 2 test tubes with 5ml LB and ampicillin were inoculated with colonies containing BBa_K177024 on pSB1A2 plasmid. The cultures were left for overnight incubation at 37°C. This time cultures were taken from sparse plate

Results:


 * Will be determined next day by cell presence in tubes

Cloning of the mgtc promoter into the pKSII+ plasmid Kamil

Tasks: <ul> <li>Transformant selection</li> </ul>

Methods: <ul> <li>White colonies that appeared after 48h of incubation were picked up and transferred to a new petri dish.</li> <li>Liquid cultures were established along the way for plasmid purification.</li> <li>Both the dish and the liquid cultures were incubated at 37&deg;C overnight. </ul>

Results: <ul> <li>All of the newly transferred colonies turned blue overnight as well and not suprisingly the purified plasmids yealded no mgtc promoter.</li> </ul>

Conclusions: <ul> <li>It is possible that the X-gal used is not of top quality which may result in the late colorisation of the colonies.</li> <li>It is also possible that the overwhelming autoligation rate is due to the fact that the SmaI enzyme has only 50% activity in 37&deg;C, a fact that was not taken into account previously.</li> </ul>