Team/CINVESTAV-IPN-UNAM MX/Metods.htm

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   <p align="center"> Rhodofactory 2012 </p>
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   <p align="center"> <strong>Rhodofactory 2012</strong> </p>
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Latest revision as of 02:00, 26 October 2012

Rho

Materials and Methods!

Biobicks

The following biobicks were recuperated from the 2009 and 2012 iGEM distribution plates.

figuram01

Circuits assembly

Plasmids were obtained by miniprep technique using the kit: Zyppy Plasmid Miniprep Kit and quantified by Nanodrop.

For the constitutive promoters (J23101, J23102, J23104, J23107, J23108, J23111 and J23115) the following enzyme digest were used: 8 hours at 37ºC:

figuram02


After enzyme digestion, a gel was made to confirm the released fragments and the band purification was performed using the kit: Gel DNA Recovery Kit.
The biobrick J54103 was extracted from the plate and amplified by PCR of 50 μl, 25 cycles of 94 ° C 0:30, 0:45 55 ° C, 72 ° C 1:15, 72 ° C 8:00 and 4 ° C Hold Infinite.
Following the amplification of the biobrick, a digestion was made for 8 hours at 37 ° C.
Once obtained the promoters and GFP, each one was ligated to GFP reporter gene, by ligation of 16 hours at 16 ° C, with a final volume of 20 μL.
Following the ligation of 16 hours, chemiocompetents TOP 10 cells were transformed by heat shock. At 12 hours of culture, the colonies were observed, the ones that were selected were those which showed green fluorescence under UV light.
Miniprep was performed to extract plasmids of selected cells, as well as the vector J04450 cells. They were digested with the enzymes EcoRI and PstI for 16 hours.
The bands were purified cutting the inserts constructions of promoters and the J04450 biobrick containing the chloramphenicol resistance.
Following this, a ligation of the promoter and the vector was made.
From colonies which showed fluorescence, a final confirmation was performed by a digestion of the extracted DNA plasmid.

Promoters Characterization

With the selected colonies, an overnight culture was made in M9 media(minimal media supplemented with 0.2% CAA). After 12 hours the culture was transferred to a 96 well plate at a 1:10 dilution (20 μl of culture and 180 μL of fresh M9 medium).
OD and fluorescence measurements of the selected colonies were performed at intervals of 30 minutes for 16 h.
From the results the PopS were calculated (polymerases per second).



 

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