Difference between revisions of "Part:BBa K1800002:Experience"

Revision as of 09:38, 29 October 2016

Applications of BBa_K1800002 BBa_ K1800002

HRP :Proof of Concept

Hairy Root Culture

In 2016 A. tumefaciens containing the BBa_ K1800002 in the pORE vector was grown in YEP medium for 16 hours at 25 Celsius. The bacteria culture was pelleted by spinning at for 10 minutes. The supernatant was poured off, and the pellet resuspended in nitrogen-free plant nutrient medium until the final value of 0.2 was achieved. Rock wool plugs, about 0.5 inches thick, were inoculated with 5mL of solution containing the resuspended pellet of A. tumefaciens was added to a hole in the wool. Now into the hole made in the wool a 3 cm stem from the sterile tobacco plants was placed, and the wool placed into petri dishes which were then placed into a clear storage container. The container is then covered with a lid and the container sat under ambient light at 25 Celsuis for 5 days under ambient light in the flow hood. After dehydration treatment, the plugs are saturated with deionized water, and the cover of the container replaced. The plugs were then checked periodically and watered when necessary for the remainder of the induction period. After two to three weeks’ hairy roots grew out of the rock wool. The hairy roots contain the horseradish peroxidase gene.
BBa_ K1800000

Chemical Assay to Test Hairy Root Transformation

To detect the horseradish peroxidase in the newly formed hairy roots, a chemical assay was performed. Into 5mL of deionized water(a piece of transformed hairy root along with 5mL of the chemical 3,3′,5,5′-Tetramethylbenzidine (TMB) were placed into a 15mL microcentrifuge tube. As a control, two other reactions were set up. A tube containing 5 mL of TMB with 5 mL of and a tube containing 5 mL of , 5mL of TMB, and a piece of untransformed root. The horseradish peroxidase will turn a pale blue color that can be read spectrophotometrically at 370 or 620-650nm (Sigma-Aldrich Co.LLC, n.d.)
https://static.igem.org/mediawiki/2016/thumb/c/c4/T--Georgia_State--HRP_control_.jpg/800px-T--Georgia_State--HRP_control_.jpg ===User Reviews=== BBa_K1800002 StartReviews BBa_K1800002 EndReviews

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 <h2 style="margin-left: 0cm; text-indent: 0cm; font-weight: bold; font-size: 24px;">HRP
 :Proof of Concept  </h2>
+<h2 style="margin-left: 0cm; text-indent: 0cm; font-weight: bold; font-size: 24px;">Hairy Root Culture</h2>
 <div style="text-align:justify;">
-The A. tumefaciens containing the BBa_ K1800002 in the pORE vector was grown in YEP medium for 16 hours at 25C. The bacteria culture was pelleted by spinning at for 10 minutes. The supernatant was poured off, and the pellet resuspended in nitrogen-free plant nutrient medium until the final  value of 0.2 was achieved.  Rock wool plugs, about 0.5 inches thick, were inoculated with 5mL of solution containing the resuspended pellet of A. tumefaciens was added to a hole in the wool. Now into the hole made in the wool a 3 cm stem from the sterile tobacco plants was placed, and the wool placed into petri dishes which were then placed into a clear storage container. The container is then covered with a lid and the container sat under ambient light at 25C for 5 days under ambient light in the flow hood. After dehydration treatment, the plugs are saturated with deionized water, and the cover of the container replaced. The plugs were then checked periodically and watered when necessary for the remainder of the induction period. After two to three weeks’ hairy roots grew out of the rock wool. The hairy roots contain the horseradish peroxidase gene.
+In 2016 A. tumefaciens containing the BBa_ K1800002 in the pORE vector was grown in YEP medium for 16 hours at 25 Celsius. The bacteria culture was pelleted by spinning at for 10 minutes. The supernatant was poured off, and the pellet resuspended in nitrogen-free plant nutrient medium until the final  value of 0.2 was achieved.  Rock wool plugs, about 0.5 inches thick, were inoculated with 5mL of solution containing the resuspended pellet of A. tumefaciens was added to a hole in the wool. Now into the hole made in the wool a 3 cm stem from the sterile tobacco plants was placed, and the wool placed into petri dishes which were then placed into a clear storage container. The container is then covered with a lid and the container sat under ambient light at 25 Celsuis for 5 days under ambient light in the flow hood. After dehydration treatment, the plugs are saturated with deionized water, and the cover of the container replaced. The plugs were then checked periodically and watered when necessary for the remainder of the induction period. After two to three weeks’ hairy roots grew out of the rock wool. The hairy roots contain the horseradish peroxidase gene.
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 <title>BBa_ K1800000</title>