Difference between revisions of "Part:BBa K1616023:Experience"

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===Applications of BBa_K1616023===
 
===Applications of BBa_K1616023===
 
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<b>Liquid culture of bacteria transformed with on <bbpart>BBa_K325909</bbpart>, <bbpart>BBa_K115900</bbpart> and <bbpart>BBa_K1616023</bbpart> 96 wells plates</b>
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<b>Liquid culture of bacteria transformed with <bbpart>BBa_K325909</bbpart>, <bbpart>BBa_K115900</bbpart> and <bbpart>BBa_K1616023</bbpart></b>
 
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Competent cells have been transformed and selected on agar plates containing chloramphenicol. Then, 96 wells plates have been inoculated with colonies to perform liquid culture.
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Light intensity has been measured 5 hours after induction with arabinose or IPTG depending on the Biobrick, when DO 600 was between 0,6 and 0,8. Bba_K325909 already has the substrate synthesis within its sequence, therefore we only had to measure the kinetic during 5h of induction and took the maximum value. For the others, we added NanoGlow substrate from Promega (ratio 1:1), measured kinetics over 120 min and took the maximum value.
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Protein production has been induced when OD<sub>600</sub> values were located between 0.6 and 0.8 with inducer such as arabinose or IPTG depanding of genetic constructions and a diminution of the temperature from 37°C to 30°C. After 5 hours of incubation, 50 µl of NanoGlow substrate (Promega) have been added to bacterial culture and light emissions have been measured during the following 120 minutes (excepted for bacteria transformed with <bbpart>BBa_K325909</bbpart> for which light emissions have been measured during all the experiment). For all colonies the maximal value has been saved.
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[[Image:Nanoluc_characterization_barchart_1.png|center|500px|thumb|'''Fig. 1:''' Relative light unit depending on the concentration of IPTG]]
 
[[Image:Nanoluc_characterization_barchart_1.png|center|500px|thumb|'''Fig. 1:''' Relative light unit depending on the concentration of IPTG]]

Revision as of 00:41, 24 September 2015

Applications of BBa_K1616023


Liquid culture of bacteria transformed with BBa_K325909, BBa_K115900 and BBa_K1616023

Light intensity has been measured 5 hours after induction with arabinose or IPTG depending on the Biobrick, when DO 600 was between 0,6 and 0,8. Bba_K325909 already has the substrate synthesis within its sequence, therefore we only had to measure the kinetic during 5h of induction and took the maximum value. For the others, we added NanoGlow substrate from Promega (ratio 1:1), measured kinetics over 120 min and took the maximum value.

Fig. 1: Relative light unit depending on the concentration of IPTG


We worked in BL21(DE3) bacteria for our plasmid “T7 Nanoluc”. Thus, addition of IPTG activates the induction of the Plac promoter and then expression of T7 RNA polymerase is possible. It results in different light intensities proportional to the concentration of IPTG: the more IPTG you have, the brighter!

Fig. 2: Comparison of the relative light unit using 3 differents Biobricks:Induction of BBa_K325909 and BBa_K1159001 with a solution of arabinose 1%, and a solution of IPTG 1 mM for T7-Nanoluc.


We compared our new T7 Nanoluc to the first Luxbrick created by Cambridge in 2010 (BBa_K325909), and to BBa_K1159001. T7 NanoLuc showed a 1,2 times brighter light intensity (RLU) than BBa_K1159001 and a light intensity approximately 140 times brighter than BBa_K325909.

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