Difference between revisions of "Part:BBa K2826008"

Revision as of 17:15, 17 October 2018

copper sensor1

A device to detect the copper ion

Sequence and Features

Usage and Biology

The device contains a copperA promoter,a riboJ,a RBS,a RFP repoter and two terminator. This device will be used to detect copper in solution. The copA promoter is active in the presence of copper. RiboJ can reliably maintain relative promoter strengths. RiboJ helps reduce the leakage of copA promoter greatly. After adding copper ions, the expression of red-fluorescent protein increased steadily.

CopA, the principal copper efflux ATPase in Escherichia coli, is induced by elevated copper in the medium.The copA promoter is Copper-responsive and regulated by CueR,a Member of the MerR Family.

Copper ion detection experiment

experimental method：

1. take 50mL centrifuge tubes and labeled them by marker pen. Each centrifuge tube was added with 2.5mL LB medium and 2.5mLSterilized water, 15 μL chloramphenicol. When measuring wastewater, replace 2.5 ml of ultrapure water with 2.5 ml of waste water.

2. Take tube inoculate 0.02OD strain (DH5a transformed with plasmid copper sensor1) in each tube, and sealed with a lid. cultured at 37 degrees on a shaker for 10 hours.

3. Take 100ul sample every two hours and store on ice.

4. all samples were added to a 96 well plate, and the corresponding OD600 value and fluorescence intensity value were read out by Tecan Infinite M200.

experimental result：

Fig.1.Changes in fluorescence induced by different thickness of copper ion in Copa.s1

Fig.2.Changes in red intensity induced by different thickness of copper ion in Copa.s1 strain after 10 hours of cultivation

The figures above show that As the concentration of copper ions in the solution increases, the fluorescence intensity becomes higher and the bacteria red deepens。The concentration of copper ions in the waste water is close to 3ml/L. The detection results are similar to measurement result of ICP.

Improvement： We concluded that The design of RiboJ after copA promoter works well, Even after replacing GFP in BBa_K2088015 with RFP. After using RFP, the detection results of the copper sensor are more conspicuous. The experimental results show that the copper sensor can reflect the true copper concentration in the solution by the red fluorescence intensity and the red intensity of the liquid. We tested the waste water in our lives and the results were in line with expectations.