Team:Warsaw/Calendar-Main/8 September 2009

Cloning of the mgtc promoter into the pSB1A3 plasmid Kamil

Tasks:  Colonies transfer Liquid cultures establishment 

Methods:  Appropriate colonies (all 5 of them) were transferred to a new plate containing ampicilin. Liquid cultures were established in 4 ml LB medium supplemented with ampicilin and incubated overnight with areation. 

Cloning of the cro-box into the pSB1A3 plasmid Kamil

Tasks:  Colonies transfer Liquid cultures establishment</li> </ul>

Methods:  Appropriate colonies (all 7 of them) were transferred to a new plate containing ampicilin.</li> Liquid cultures were established in 4 ml LB medium supplemented with ampicilin and incubated overnight with areation.</li> </ul>

Assembly of endosomal detection operon Marcin

Task 1: Isolation of following construct from liquid bacterial culture: Methods:
 * BBa_K177037 on pSB1A3 plasmid
 * Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here

Task 2: Methods: 2 &mu;l purified plasmid DNA product 1 &mu;l PstI (Fermentas) 1 &mu;l XbaI (Fermentas) 2 &mu;l Buffer Tango (Fermentas) 15 &mu;l MQ water Results:
 * Digest previously isolated plasmid to verify the correctness of the ligation:
 * Reaction mixture composition:
 * Digest was performed 3 hours

Task 3: Methods: 1. μl purified plasmid DNA product 1 μl XbaI (Fermentas) 1 μl PstI (Fermentas) 2 μl Buffer Tango (Fermentas) 15 μl MQ water
 * Digest of following constructs ( both of them are on pSB1A3 plasmid):
 * BBa_K177035
 * BBa_K177036
 * Reaction mixture composition:
 * Digestions were carry out 5 hours and subsequently were inactivated via heating for 20 minutes

Comment: The length of digested sequence is unexpected. The correct sequence should have had 1500 bp. Althought in each case the length is approximately 950 bp. It is obligated to carry out sequencing reaction to reveal the identity of this DNA.

Assembly of fusion proteins Marcin

Task:1 Construct to transform: Methods:
 * Transformation of chemocompetent E. coli TOP10 strain
 * signal peptid from Cox1 CDS on pKSII vector
 * Ligation mixture was thermally inactivated in 65 &deg;C
 * Detailed protocol of transformation is described here

Acquiring the internaline A gene from Listeria monocytogenes st. EDG-e genome
Jarek

Tasks: Inverting PLacI regulated parts of the switch and assembling the switch on a single plasmid Ania Tasks:   </li> </ul>
 * Phosphorylation of PCR product with T4 phage kinase.
 * Digestion of pUC18 with HincII endonuclease and dephosphorylation of it's ends with CIAP phosphatase.
 * Seting ligation of internaline A with pUC18.