Team:Warsaw/Calendar-Main/16 September 2009

Cloning of the mgtc promoter into the pSB1A3 plasmid Kamil

Tasks:  pSB1A3 plasmid digest 

Methods:  The digest mix was prepared as follows: 5&mu;l concentrated plasmid 1&mu;l Buffer O (Fermentas) 0,5&mu;l EcoRI enzyme 0,5&mu;l PstI enzyme 3&mu;l H2O  The digest was carried out in 37&deg;C overnight and inactivated for 15 min. in 80&deg;C. The product was later purified using the Montage PCR Centrifugal Filter Device. 

Cloning of the cro-box into the pSB1A3 plasmid Kamil

Tasks:  pSB1A3 plasmid digest 

Methods:  The digest mix was prepared as follows: 5&mu;l concentrated plasmid 1&mu;l Tango Buffer (Fermentas) 0,5&mu;l XbaI enzyme 0,5&mu;l PstI enzyme 3&mu;l H2O </li> The digest was carried out in 37&deg;C overnight and inactivated for 15 min. in 80&deg;C.</li> The product was later purified using the Montage PCR Centrifugal Filter Device.</li> </ul>

Assembly of fusion proteins Marcin

Task 1:

Construct to transform: Methods:
 * Transformation of chemocompetent E. coli TOP10 strain
 * mitochondrial signal peptide CDS on pSB1A3 plasmid
 * Detailed protocol of transformation is described here

Assembly of endosome detection module: Marcin

Task 1: Prepare of BBa_K177044

Comment:

Due to some difficulties with preparing the BBa_K177037 I decided to create another biobrick, BBa_K177044 compose with following biobricks: Methods: 20 &mu;l purified plasmid DNA product 1 &mu;l XbaI (Fermentas), or 0.7 &mu; SpeI (Fermentas), the latter one was used to cut BBa_K177035 1 &mu;l PstI (Fermentas) 5 &mu;l Buffer Tango (Fermentas) 23 &mu;l MQ water
 * BBa_K177035
 * BBa_K177043
 * Restriction digest of previously denoted biobricks:
 * Reaction mixture composition:
 * Reactions were carried out 6 hours and were subsequently thermally inactivated.

Task 2: Gel-out of the previously described, digested biobricks

Methods:
 * All gel-outs were performed using the EurX gel-out kit according to the manual

Results:

Task 3: Ligation of the BBa_K177044 Methods: 10 μl digested insert 8 μl digested vector 2 μl Tango buffer(Fermentas) 3 μl dNTPs mixture (EurX, concentration 5 mM) 1 μl ligase T4 (Fermentas)
 * Ligation mixture composition:
 * Ligation were carried out 16 hours