Difference between revisions of "Part:BBa K1555000:Experience"

Revision as of 06:41, 21 October 2016

This sensor can be activated in low copper concentration and it's truely useful.

2h 0mg/L copper ion 2h 1mg/L copper ion

Applications of BBa_K1555000

This year we add the RiboJ(a section of functional RNA used to buffer synthetic circuits from genetic context) between the copA pomoter and the RBS(B0030) as an improvement.Also we used GFP as the reporter( BBa_K2088015 copA+riboJ+B0030+GFP+B0015).For more details please check BBa_K2088015.

Experience of NAU-CHINA-2016 iGEM team--Kejian Shi

Experimental Design

Overview

CopA, the principal copper effluxATPase in Escherichia coli, is induced by elevated copper in the medium.[1] CopA promoter is active in the presence of copper ion.

First experiment:

We test copA promoter in BL21（DE3）,DH5α. By measuring fluorescence intensity in cells by flow cytometer,we got data to analyze sensitivity and specificity of copA promoter.

Results:

In our experiment, copA promoter was induced by different concentration of copper ion (37.5umol/L、50umol/L、62.5umol/L、75umol/L) . That fluorescence intensity in cell increase firstly and decreasewith small oscillations.（Fig.3A,B） At 4-5th hour fluorescence intensity in cell increases dramatically. Dose response curves was fitted to twice induction within 9 hours. CopA promoter has relative leaky basal expression by comparing the negative control’s output and basal leakage of copA promoter in E. coli expression systems(Fig.4). In comparison of two graphs A、B, we can obviously find that the degradation of protein is much faster in DH5α than that in BL21（DE3），because BL21（DE3 ） has a deficiency of protease. In the group of 0μmol/L Cu2+, the fluorescence shows a trend of falling firstly then rising（Fig.3C）. Actually, the fluorescence which produced by the leakage of copA will not change. The change quantity comes from the different growth periods of the E.coli. We added 40ul bacterial fluid into new medium with inductionto start measuring. So bacteria will go through a period of growing from growth period to maturation period, so as to the change of the fluorescence.Maturation period is great period for the expression of protein.

Fig.3.A.Changes in fluorescenceintensity induced by different concentration of copper ion in E.coli BL21 (DE3). B.Changes in fluorescence intensityinduced by differentconcentration of copper ion in E.coli DH5α. C .Changes in fluorescenceintensity in E.coli without induction comparing with two strains. D .Changes in fluorescenceintensity in E.coli which do not contain copA promoter comparing with two strains.

Fig.4.CopA promoter has leaky basal expression

Second experiment:

We placed insulator RiboJ between copA promoter and RBS.(Fig.5)

Fig.5.Two device used to detect copper in solution, the upper device has riboJ and the under one has no riboJ.

Result:

We use 50μmol/L copper ion to induce copA promoter.A device without RiboJ has an unstable Fluorescent quantity. At fourth hour, the fluorescence intensity in cells rose sharply. By contrast,a device with RiboJ response to copper ion and express GFP gradually. (Fig 6A) In addition,a device without RiboJ has high leakage with fluctuation. However, a device with RiboJ has low and stable leakage. (Fig 6B)

Fig .6 A.Changes in fluorescenceintensity induced by copper ion in E.coli over time B.leakage of copA promoter over time

Discussion: We proposed that RiboJ helps reduce and control leakage of copA promoter.

Reference: [1] Outten FW, Outten CE, Hale J, O'Halloran TV. Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal MerR homologue, cueR. Journal of Biological Chemistry 2000;275:31024-9.

User Reviews

UNIQ128dd6daec837642-partinfo-00000000-QINU

•••••

HSiTAIWAN

Detecting Copper in Chinese medicine

Objective

Measure the E. coli fluorescence intensity in different copper ion concentration with 3μg/ml Chinese medicine.

Methods

Copper ion solution with different copper ion concentration (0, 3, 9, 27ppm) is added into E. coli DH5α with Plasmids encoding GFP under copper promoter(part: BBa_K1555000).
Fluorescence was measured at:

Excitation peak: 470nm
Emission peak: 508nm

every 30 minutes.

Results

This part is still specific to copper in Chinese medicine, so we can apply it in Chinese medicine examination.

Discussion

Since E. coli expresses stronger fluorescence in higher copper concentration, this is a relevant promoter to detect the existence and measure the amount of copper.

Review

This part works great! HSiTAIWAN iGEM team 2016 used this part to detect copper ions in Chinese Medicine. The results were as expected. The reporter performed well without being interfered by complex substances. This part performed in an expected pattern and is an useful reporter.

UNIQ128dd6daec837642-partinfo-00000002-QINU

@@ Line 25: / Line 25: @@
 In our experiment, copA promoter was induced by different concentration of copper ion (37.5umol/L、50umol/L、62.5umol/L、75umol/L) . That fluorescence intensity in cell increase firstly and decreasewith small oscillations.（Fig.3A,B） At 4-5th hour fluorescence intensity in cell increases dramatically. Dose response curves was fitted to twice induction within 9 hours. CopA promoter has relative leaky basal expression by comparing the negative control’s output and basal leakage of copA promoter in E. coli expression systems(Fig.4). In comparison of two graphs A、B, we can obviously find that the degradation of protein is much faster in DH5α than that in BL21（DE3），because BL21（DE3 ） has a deficiency of protease. In the group of 0μmol/L Cu2+, the fluorescence shows a trend of falling firstly then rising（Fig.3C）. Actually, the fluorescence which produced by the leakage of copA will not change. The change quantity comes from the different growth periods of the E.coli. We added 40ul bacterial fluid into new medium with inductionto start measuring. So bacteria will go through a period of growing from growth period to maturation period, so as to the change of the fluorescence.Maturation period is great period for the expression of protein.
 [[Image:NAUCHINAdescription_fig3.png|800px|thumb|centre|Fig.3.A.Changes in fluorescenceintensity induced by different concentration of copper ion in E.coli BL21 (DE3). B.Changes in fluorescence intensityinduced by differentconcentration of copper ion in E.coli DH5α. C .Changes in fluorescenceintensity in E.coli without induction comparing with two strains. D .Changes in fluorescenceintensity in E.coli which do not contain copA promoter comparing with two strains.]]
+[[Image:NAUCHINAdescription_fig4.png|800px|thumb|centre|Fig.4.CopA promoter has leaky basal expression]]
 Second experiment:
-We placed insulator RiboJ between copA promoter and RBS.
+We placed insulator RiboJ between copA promoter and RBS.(Fig.5)
+[[Image:NAUCHINAdescription_fig5.png|800px|thumb|centre|Fig.5.Two device used to detect copper in solution, the upper device has riboJ and the under one has no riboJ. ]]
 Result:
 We use 50μmol/L copper ion to induce copA promoter.A device without RiboJ has an unstable Fluorescent quantity. At fourth hour, the fluorescence intensity in cells rose sharply. By contrast,a device with RiboJ response to copper ion and express GFP gradually. (Fig 6A) In addition,a device without RiboJ has high leakage with fluctuation. However, a device with RiboJ has low and stable leakage. (Fig 6B)
+[[Image:NAUCHINAdescription_fig6.png|800px|thumb|centre|Fig .6 A.Changes in fluorescenceintensity induced by copper ion in E.coli over time  B.leakage of copA promoter over time]]