Difference between revisions of "Team:NAU-CHINA/Experiment/MHBPT"
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| − | <p>We found that the promoter is induced by 3-hydroxybenzoate, since the fluorescent quantity see a significantly rise with the increase of concentration of 3-hydroxybenzoate. We supposed that, the bacteria is in logarithmic growth phase before the third hour when the speed of GFP production is slower than that of protein breakdown. At about the third hour, the GFP in solution of 4000μmol/L and 5000μmol/L 3-hydroxybenzoate is beginning to increase. On the contrary, GFP in solution without 3-hydroxybenzoate is still reducing. We infer that the bacteria is in maturation period at about the third hour when cell is beginning to accumulate the protein. At the same time, the increasing of GFP in solution without 3-HBA comes from the leakage of the promoter Mhbpt which can not balance the protein degradation. The fluorescent quantity in solution of 5000μmol/L of 3-HBA is nearly | + | <p>We found that the promoter is induced by 3-hydroxybenzoate, since the fluorescent quantity see a significantly rise with the increase of concentration of 3-hydroxybenzoate. We supposed that, the bacteria is in logarithmic growth phase before the third hour when the speed of GFP production is slower than that of protein breakdown. At about the third hour, the GFP in solution of 4000μmol/L and 5000μmol/L 3-hydroxybenzoate is beginning to increase. On the contrary, GFP in solution without 3-hydroxybenzoate is still reducing. We infer that the bacteria is in maturation period at about the third hour when cell is beginning to accumulate the protein. At the same time, the increasing of GFP in solution without 3-HBA comes from the leakage of the promoter Mhbpt which can not balance the protein degradation. The fluorescent quantity in solution of 5000μmol/L of 3-HBA is nearly twice of fluorescent quantity in solution without 3-HBA. So that the difference between the two groups cannot be seemed as error. In conclusion, we found obvious induction of Mhbpt by 3-HBA.</p> |
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<img src="https://static.igem.org/mediawiki/2016/6/6d/T--NAU-CHINA--MHBPT04.png"> | <img src="https://static.igem.org/mediawiki/2016/6/6d/T--NAU-CHINA--MHBPT04.png"> | ||
Latest revision as of 19:20, 19 October 2016
Mhbpt promoter
Introduction
Mhbpt promoter Fig.1. is a composite part which contains two promoters. This sequence got from mhb structural genes. The promoters of mhbR and the mhb operon ares70-type and overlap with each other. The fragment Fig.2. we got contains the necessary element for regulator binding and catabolic pathway induction. It demonstrated that site 1 is a critical binding site for MhbR regulation .3-hydroxybenzoate is one important factor of the induction.
Fig. 1. Mhbpt promoter
Fig.2.Overview of the promoter–operator regions ofmhbRandmhboperon
Experiment
To test the induction of 3-HBA to promoter Mhbpt, we used 3-hydroxybenzoate with different concentration to induce the Mhbpt promoter, then tested the fluorescent quantity to analyze the level of induction using flow cytometer.
Fig.3.A device which can report the presence of 3-hydroxybenzoate in environment
Results
We found that the promoter is induced by 3-hydroxybenzoate, since the fluorescent quantity see a significantly rise with the increase of concentration of 3-hydroxybenzoate. We supposed that, the bacteria is in logarithmic growth phase before the third hour when the speed of GFP production is slower than that of protein breakdown. At about the third hour, the GFP in solution of 4000μmol/L and 5000μmol/L 3-hydroxybenzoate is beginning to increase. On the contrary, GFP in solution without 3-hydroxybenzoate is still reducing. We infer that the bacteria is in maturation period at about the third hour when cell is beginning to accumulate the protein. At the same time, the increasing of GFP in solution without 3-HBA comes from the leakage of the promoter Mhbpt which can not balance the protein degradation. The fluorescent quantity in solution of 5000μmol/L of 3-HBA is nearly twice of fluorescent quantity in solution without 3-HBA. So that the difference between the two groups cannot be seemed as error. In conclusion, we found obvious induction of Mhbpt by 3-HBA.
Fig.4.The abscissa is the time of induction,the ordinate represents the fluorescent intensity.
Reference
[1] Lin L X, Liu H, Zhou N Y. MhbR, a LysR-type regulator involved in 3-hydroxybenzoate catabolism via gentisate in Klebsiella pneumoniae M5a1[J]. Microbiological research, 2010, 165(1): 66-74.
