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

Methods:

• Appropriate colonies (all 7 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.

Assembly of endosomal detection operon

Marcin

Task 1: Isolation of following construct from liquid bacterial culture:

• [http://partsregistry.org/Part:BBa_B0032BBa_B0032] with [http://partsregistry.org/Part:BBa_E0022BBa_E0022] connected to [http://partsregistry.org/Part:BBa_B0032BBa_B0032] with [http://partsregistry.org/Part:BBa_C0051BBa_C0051] on [http://partsregistry.org/Part:pSB1A3pSB1A3] plasmid

Methods:

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

Task 2:

• Digest previously isolated plasmid to verify the correctness of the ligation:

Methods:

• Reaction mixture composition:

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

• Digest was performed 3 hours

Results:

Task 3:

• Digest of folowing constructs ( both of them are on [http://partsregistry.org/Part:pSB1A3pSB1A3] plasmid):
  • [http://partsregistry.org/Part:BBa_B0032BBa_B0032] with [http://partsregistry.org/Part:BBa_E0022BBa_E0022]
  • [http://partsregistry.org/Part:BBa_B0032BBa_B0032] with [http://partsregistry.org/Part:BBa_C0051BBa_C00251]

Methods:

• Reaction mixture composition:

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

• 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

• Transformation of chemocompetent E. coli TOP10 strain

Construct to transform:

• signal peptid from Cox1 CDS on pKSII vector

Methods:

• Ligation mixture was thermally inactivated in 65 °C
• Detailed protocol of transformation is described here

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

Jarek

Tasks:

• 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.