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Team:SDU-Denmark/Tour52
From 2013.igem.org
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| - | <a class="popupImg alignRight" style="width:200px" href="https://static.igem.org/mediawiki/2013/8/88/SDU2013_Characterization_AraCPara_1.png" title="A) Normalized intensity of HRT2 mRNA using intensity of 5S rRNA as reference. Duplicates of MG1655 strains carrying either pSB1C3-Para-HRT2 (BBa_K1088024) (-araC) or pSB1C3-Pcon-araC-term-Para-HRT2 (BBa_K1088016) (+araC) were induced with 0.2 % arabinose at time t=0 min (OD600=0.8), and samples were taken at times: -2 min, 15 min, and 30 min. The normalized intensity of sample -araC -2min were set to 1 and the other samples are relative to that. Within 15 min of induction the expression levels are at its maximum in both strains, and overexpression of AraC does not seem to be necessary for expression control of the arabinose promoter. | + | <a class="popupImg alignRight" style="width:200px" href="https://static.igem.org/mediawiki/2013/8/88/SDU2013_Characterization_AraCPara_1.png" title=" |
| - | B) Northern blot result reflecting diagram."> | + | |
| + | A) Normalized intensity of HRT2 mRNA using intensity of 5S rRNA as reference. Duplicates of MG1655 strains carrying either pSB1C3-Para-HRT2 (BBa_K1088024) (-araC) or pSB1C3-Pcon-araC-term-Para-HRT2 (BBa_K1088016) (+araC) were induced with 0.2 % arabinose at time t=0 min (OD600=0.8), and samples were taken at times: -2 min, 15 min, and 30 min. The normalized intensity of sample -araC -2min were set to 1 and the other samples are relative to that. Within 15 min of induction the expression levels are at its maximum in both strains, and overexpression of AraC does not seem to be necessary for expression control of the arabinose promoter. | ||
| + | B) Northern blot result reflecting diagram. | ||
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<img src="https://static.igem.org/mediawiki/2013/8/88/SDU2013_Characterization_AraCPara_1.png" style="width:200px" /> | <img src="https://static.igem.org/mediawiki/2013/8/88/SDU2013_Characterization_AraCPara_1.png" style="width:200px" /> | ||
Figure X. | Figure X. | ||
Revision as of 01:52, 4 October 2013
Characterization
“And by what, O Socrates, is the soul nourished?” - Hippocrates
“By knowledge, of course, I said.” - Socrates
Characterization of our biobricks is a chance for us to prove that our design works as intended. We invite you along on a journey through our attempts to obtain proof of concept; to show that Bacteriorganic Rubber is a true possibility. This page will slowly guide you through our results, but keep in mind that not everything is presented below. For all the details, consult our protocol page, where you will find a comprehensive picture of our project.
Characterization of LacI/Plac
Characterization of AraC/Para
Figure X.
The HRT2 gene is under the control of the arabinose promoter (which is arabinose inducible). Therefore we assayed if we are in fact capable of inducing the expression by addition of arabinose, and that expression is shut down prior to induction. The assay was carried out by measuring the mRNA levels of HRT2 using the northern blotting technique.
To test if overexpression of AraC improved expression control, devices with and without the araC device was assayed. Duplicates of MG1655 strains carrying either pSB1C3-Para-HRT2 or pSB1C3-Pcon-araC-term-Para-HRT2 were grown to late-exponential phase: OD600=0.8. At this OD the strains were induced with 0.2 % arabinose at time t=0 min, and samples were taken at times: -2 min, 15 min, and 30 min. Total RNA purified from the samples were run on a gel, blotted onto a membrane, and hybridized with probes specific for HRT2 mRNA and 5S rRNA (loading control), respectively.
The results proves that we are capable of inducing our HRT2 devices with arabinose. There is only little expression before induction and within the first 15 min the expression is at its maximum. Overexpression of AraC does not seem to have any effect on the expression levels after 15 min compared to natural levels of AraC, though we cannot conclude that it has no effect in the minutes prior to 15 min after induction (Fig. X).
Our obtained experience has been added to the experience of the part encoding the arabinose promoter on parts registry.
Characterization of dxs (B. subtilis)
