Team:Warsaw/Calendar-Main/15 July 2009

Creating devices to test promoters in E. coli strains (devices BBa_K177024 and BBa_K177025)
Franek

Tasks:


 * Selecting clones with BBa_K177024 and BBa_K177025

Methods:


 * Tranformants with BBa_K177024 and BBa_K177025 clones were recognised due to the fluorescence under UV light.


 * 4 positive BBa_K177024, and 16 BBa_K177025 were found.


 * Each clone was transferred to new plate with LB, agar-agar and IPTG or 0.2 % arabinose. Liquid cultures were also established.

Results:


 * BBa_K177024 and BBa_K177025 were successfully created!

Cloning of the mgtc promoter into the pKSII+ plasmid Kamil

Tasks:  Bacteria transformation 

Methods:  A 200&mu;l batch of chemocompetent bacteria was transformed with 15&mu;l of ligation mix and incubated overnight on petri dishes

containing LB medium supplemented with ampicilin, X-gal and IPTG. 

Results: The transformation yielded only a couple of blue colonies.

Conclusions:  The transformation needs to be redone, this time using more of the ligation mix. 

Assembly of endosomal detection operon Marcin Task 1:  Isolate plasmids containing the biobricks  Methods:  Plasmids weren 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</a>.</li> After the isolation 1 ul of each plasmid solution was diluted to 10 ul and loaded into the 1% agarose gel</li> Electrophoresis condition:</li> </ul> voltage - 70V time - 30 min  Next the gel was photographed:</li> </ul> <img src="http://2009.igem.org/wiki/images/4/41/Biobricks_izolacja_15_07_09.png" width="60%" height="60%"> Samples of plasmids after isolation Legend: 1-3 - <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> 4-6 - <a href="http://partsregistry.org/Part:BBa_C0040"> BBa_c0040</a> 7-9 - <a href="http://partsregistry.org/Part:BBa_C0051"> BBa_C0051</a> 10-12 - <a href="http://partsregistry.org/Part:BBa_E0032"> BBa_E0032</a> Comment: Isolation of plasmids was successful. Cloning of p53 coding sequence Marcin

Task:  Cloning p53 coding sequence to pKS plasmid</li> </ul> Methods:  Ligation mixture composition: 14 &mu;l digested p53 1.5 &mu;l digested pKS 5 &mu;l ligation buffer (Invitrogen) 1 &mu;l ligase T4 </li> Duration of ligation was about 20 hours; reaction was conducted in 16 &deg;C (approximately).</li> </ul>

Insertion of the pho gene into the pKSII+ plasmid Kama  Isolation of plasmid containing an insert was performed with the <a href=http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf>A&A plasmid mini kit</a></li> </ul> <img src="http://2009.igem.org/wiki/images/8/87/2009_07_16_pKS_pho_opisany.JPG"> Notes  All samples contain empty plasmid</li> </ul> Construction of K177012 operon1_part2

Ania

Tasks:
 * Electrophoresis, gel extraction and ligation of:
 * BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid (XbaI/PstI = prospective insert)
 * BBa_B0032 - RBS.3 on the pSB1A2 ampicillin resistant plasmid (SpeI/PstI = prospective vector)