Assembly of endosome detection operon

Marcin

Task 1: Isolation of pSB1A3 plasmid containing BBa_K177044

Methods:

• Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here

Task 2: Restriction digest of previously isolated plasmids:

Methods:

• Reaction mixture composition:

3 μl purified plasmid DNA product
1 μl XbaI (Fermentas)
1 μl PstI (Fermentas) 
2 μl Buffer Tango (Fermentas)
13 μl MQ water

• Reactions were carried out 2 hour

Results:

Some sequences 1500 bp length are visible on the gel but I need to determine whether it is expected product or contamination (bacteria containing plasmid with listeriolysin were also presented on the same plate)

Task 3: PCR reaction to determine the identity of isolated (Task 1) DNA

Commment:

I decided to perform PCR reaction using primers for listeriolysin. If the plasmids contain BBa_K177044 there will be no PCR product.

Methods:

• Reaction mixture composition:

0.1 μl purified plasmid DNA product
1.4 μl Opti Taq Polymerase (EURx)
1 μl dNTPs (EURx)
2 μl Opti Taq Buffer (Fermentas)
15.5 μl MQ water

Results:

All isolated plasmid contain listeriolysin. It is necessary to carry out ligation one more time

Task 4:

• Transformation of chemocompetent E. coli TOP10 strain

Construct to transform:

• BBa_K177044

Methods:

• Ligation mixture was thermally inactivated in 80 °C for 20 minutes
• Detailed protocol of transformation is described here

Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil

Tasks:

• Selection of the colonies

Methods:

• The appropriate colonies were transferred to a fresh agarose plate and liquid cultures were established.
• The plate and the liquid cultures were incubated overnight in 37°C

Cloning of the cro-box into the pSB1A3 plasmid

Kamil

Tasks:

• Selection of the colonies

Methods:

• The appropriate colonies were transferred to a fresh agarose plate and liquid cultures were established.
• The plate and the liquid cultures were incubated overnight in 37°C