Insertion of the pho gene into the pKSII+ plasmid

Kama

Tasks:

• Isolation of fragment of the correct lenght (∼2200bp) from the gel was performed with the [http://aabiot.com/products/dna_purification/dna_fragments/gel_out/protocol_gel_out.pdf A&A "Gel-out" kit].

• purified inserts of biggest concentration were taken to next steps of cloning (marked with an arrow, ~30ng/μl)

Cloning of p53 coding sequence

Marcin

Task 1:

• Breed bacteria to isolate plasmid containing p53 coding sequence

Methods:

1. Prepare LB medium with kanamycin
2. Add 3.5 ml of the medium to the probes
3. Add one bacterial colony to each probe
4. Breed the bacteria about 7 hours

Task 2:

• Isolate the plasmids from bacterial cultures and verify the effectivity of the isolation via gel electrophoresis

Methods:

• Plasmids weren isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described [http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf here]
• After the isolation 1 ul of each plasmid solution was diluted to 10 ul and loaded into the 1% agarose gel
• Electrophoresis condition:

voltage - 70V

time - 30 min

• Next the gel was photographed:

plasmid samples on the gel

Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2

Ania

Tasks:

• Transformation of chemocompetent strain of E. with [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid taken out of the distribution 200 Kit Plate 1 well 2O.

• Alkaline lysis of bacterial cultures to obtain plasmids containing following bricks:

[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0051 BBa_R0051] - promoter (lambda cI regulated); [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032] - RBS.3 (medium)

• Digestion to confirm plasmid extraction. DNA that should contain R0051 on pSB1A2 plasmid was digested with PvuI and HindII. Digestion mix contained 5µl of extracted DNA, 2µl of Fermentas BamHI buffer , 0.3µl of each enzyme and water added to obtain 20µl total volume.

• obtained fragments match expected

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:

• Alkaline lysis of bacterial cultures to obtain plasmids containing [http://partsregistry.org/Part:BBa_R0010 BBa_R0010 - lacI regulated promoter] and [http://partsregistry.org/Part:BBa_R0080 BBa_R0080 - AraC regulated promoter] bricks

• Digestion to confirm plasmid extraction.

Methods:

• 3 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing [http://partsregistry.org/Part:BBa_R0010 placI] or [http://partsregistry.org/Part:BBa_R0080 pAraC] bricks on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid. The cultures were incubated overnight at 37°C. Alkaline lysis was performed on both cultures, according to our standard procedure. The pellet from 5 ml of bacteria was used.

• DNA that should contain [http://partsregistry.org/Part:BBa_R0080 pAraC] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid was digested with BamHI and PvuI, [http://partsregistry.org/Part:BBa_R0010 placI] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] was digested with IPvuI and PvuII . Digestion mix contained 5µl of extracted DNA, 2µl of Fermentas BamHI buffer, 0.3µl of each enzyme and water added to obtain 20µl total volume. All mixes were incubated for 2h at 37°C.

Results:

• obtained fragments match expected

Making of RBS-cI part

Jarek

Tasks:

• Transformation of ligation from previous day into competent E.coli strain Top10

• Plating of transformated culture on the LB+Amp plates and incubation in 37C degree.

Evaluating the quality of bacterial medium

Andrzej

Tasks:

• Transformation of E. coli strain DH5alpha with pKs vector as a positive control

Methods:

• If you want to see detailed procedure go here
• Bacteria was plated on the medium containing kanamycin

Franek lub Monika opisze minilizy dla Moniki