Creating devices to test promoters in E. coli strains (devices [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025])

Franek

Tasks:

• Transformation of competent cells with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025]

Methods:

• Chemocompetent E. coli cells from Top10 strain were transformed according to our standard procedure with either [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] or [http://partsregistry.org/Part:BBa_K177025 BBa_K177025]

• Tranformants with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] were plated on 18 plates with LB, agar-agar and IPTG

• Tranformants with [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] were plated on 18 plates with LB, agar-agar and 0.2 % arabinose.

Results:

• Positive selection will be made according to fluorescence under UV light

Cloning p53 coding sequence

Marcin

Task 1:

• Transformation of chemocompetent strain of E. coli by plasmid containing p53 sequence

Procedure:

• The protocol of transformation has not been changed, except of giving 10 µl of ligated plasmid solution to the bacteria. If you want to see detailed procedure go here
• Bacteria was plated on the medium containing ampicillin, X-Gal and IPTG

Task 2: Restriction digest of pKS vector for future aplications:

Methods:

• Reaction mixture composition: 2 μl pKS plasmid, 1 μl XbaI (Fermentas), 1 μl SmaI (Fermentas) 2 μl Buffer Tango (Fermentas), 14 μl MQ water

Program:

digest:

1. 37°C - 8 hours
2. 65°C - 15 minutes

Assembly of endosomal detection operon

Marcin

Task 1:

• Breed bacteria to isolate plasmid containing biobricks essential for build the operon:

[http://partsregistry.org/Part:BBa_B0032 BBa_B0032]

[http://partsregistry.org/Part:BBa_C0040 BBa_C0040]

[http://partsregistry.org/Part:BBa_C0051 BBa_C0051]

[http://partsregistry.org/Part:BBa_E0021 BBa_E0021]

Methods:

1. Prepare LB medium with ampicillin
2. Add 3.5 ml of the medium to the probes
3. Add one bacterial colony to each probe
4. Breed the bacteria about 10 hours