Team:Warsaw/Calendar-Main/11 July 2009

Insertion of the pho gene into the pKSII+ plasmid
Kama

Tasks:


 * Isolation of fragment of the correct lenght (&sim;2200bp) from the gel was performed with the A&A "Gel-out" kit.

http://2009.igem.org/wiki/images/0/08/2009_07_12_pho_after_gelout_opisana.JPG


 * purified inserts of biggest concentration were taken to next steps of cloning (marked with an arrow, ~30ng/&mu;l)

Cloning of p53 coding sequence

Marcin

Task 1: Methods: Task 2: Methods:
 * Breed bacteria to isolate plasmid containing p53 coding sequence
 * 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
 * Isolate the plasmids from bacterial cultures and verify the effectivity of the isolation via gel electrophoresis
 * Plasmids weren isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described 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:



Construction of K177012 operon1_part2
Ania

Tasks:


 * Transformation of chemocompetent strain of E. with BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid taken out of the distribution 200 Kit Plate 1 well 2O.

BBa_R0051 - promoter (lambda cI regulated); BBa_B0032 - RBS.3 (medium)
 * Alkaline lysis of bacterial cultures to obtain plasmids containing following bricks:


 * Digestion to confirm plasmid extraction. DNA that should contain R0051 on pSB1A2 plasmid was digested with PvuI and HindII. Digestion mix contained 5&micro;l of extracted DNA, 2&micro;l of Fermentas BamHI buffer, 0.3&micro;l of each enzyme and water added to obtain 20&micro;l total volume.
 * obtained fragments match expected

Creating devices to test promoters in E. coli strains (devices BBa_K177024 and BBa_K177025)
Franek

Tasks:


 * Alkaline lysis of bacterial cultures to obtain plasmids containing BBa_R0010 - lacI regulated promoter and BBa_R0080 - AraC regulated promoter bricks

Methods:
 * Digestion to confirm plasmid extraction.


 * 3 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing placI or pAraC bricks on 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.

Results:
 * DNA that should contain pAraC on pSB1A2 plasmid was digested with BamHI and PvuI, placI on pSB1A2 was digested with IPvuI and PvuII . Digestion mix contained 5&micro;l of extracted DNA, 2&micro;l of Fermentas BamHI buffer, 0.3&micro;l of each enzyme and water added to obtain 20&micro;l total volume. All mixes were incubated for 2h at 37&deg;C.


 * 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: Methods:
 * Transformation of E. coli strain DH5alpha with pKs vector as a positive control
 * If you want to see detailed procedure go here
 * Bacteria was plated on the medium containing kanamycin