Difference between revisions of "Part:BBa K2826008"
 
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===Usage and Biology===
 
===Usage and Biology===
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This part is an improvement of part BBa_K2088015 </https://parts.igem.org/Part:BBa_K2088015>, which consisted by the same copper ion detecting promoter and GFP as reporter. Comparing with BBa_K2088015, our improvement is using RFP to replace GFP as reporter.
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 +
'''Improvement'''：
 +
This design modification is based on three reasons and has significant advantage:
 +
 +
1) Comparing with GFP, RFP has a visible color which can be observed by naked eyes without UV excitation, therefore, in same condition, our new part can be used for qualitatively easily. Moreover, since it is not necessary to have a UV light source for qualitatively detecting, the prototype device could be smaller and cheaper.
 +
 +
2) RFP fluoresces red-orange when excited, therefore, in case of quantitative use, it will be same as GFP.
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 +
3) Usually, red color means “dangerous” and green color means “safe”. In the case of heavy metal pollution detection, the detecting result means more “dangerous” and “alert”, thus, using red color instead of green is in keeping with common sense.
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 +
 
The device contains a copperA promoter,a riboJ,a RBS,a RFP repoter and two terminator.
 
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.
 
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.
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'''experimental method'''：
 
'''experimental method'''：
  
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2.5 ml of LB solution was added with 2.5 ml of sterilized ultrapure water, and BBa_K2826013 broth was added to make a final concentration of 0.02 OD,cultured at 37 degrees on a shaker for 10 hours. 100 microliters of bacterial solution was taken every 2 hours to detect red fluorescence. When measuring wastewater, replace 2.5 ml of ultrapure water with 2.5 ml of waste water.
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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.
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 +
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.
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 +
'''experimental result'''：
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[[Image:Copa.s1.png|600px|thumb|centre|Fig.1.Curve of REP fluorescence intensity and culture time under different copper concentrations]]
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[[Image:Copa.s1.red.png|600px|thumb|centre|Fig.2.Photos of E. Coli DH5α after culturing for 10 hours under different copper concentrations.]]
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As shown in Figure 1, with the extension of culture time, the fluorescence intensity of eRFP gradually increased which indicates that the expression level of eRFP in the medium gradually increased. In other words, the copper detection function of this working system was working normally. The concentration of copper ions in the waste water is close to 3ml/L. The detection results are similar to measurement result of ICP. Besides, as shown in Figure 2, as the copper concentration increased, the color turned redder. It is proved that the copper detection function of this working system was working as we expected.
 +
 
 +
== This part have been improved by 2019 iGEM team ASTWS-China ==
 +
 
 +
This part is improved by 2019 iGEM team ASTWS-China. To solve the problems of spreading genetic-engineered strains in the environment, we designed the improved system which could lead the strains death when IPTG is added to the culture medium. We added a composite part consisted of a lacI regulated promoter (BBa_R0010) and a lysis gene (BBa_K117000) following the existed part. The improved part has been registered with number BBa_K3152008(https://parts.igem.org/Part:BBa_K3152008). More details can be seen on https://2019.igem.org/Team:ASTWS-China/Improve.
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== This part have been improved by 2019 iGEM team Worldshaper-Shanghai ==
  
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[[Image:Copa.s1.png|800px|thumb|centre|Fig.1.Changes in fluorescence induced by different thickness of copper ion in Copa.s1]]
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This part is improved by 2019 iGEM team Worldshaper-Shanghai.
 +
To impove the problem of background leakage, the BBa_K3311008 was inserted between copA promoter and Riboj. The improved part has been registered with number BBa_K3311009 https://parts.igem.org/Part:BBa_K3311009. More details can be seen on https://2019.igem.org/Team:Worldshaper-Shanghai/Improve.

Latest revision as of 02:14, 22 October 2019


copper sensor1

A device to detect the copper ion

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 898
    Illegal AgeI site found at 1010
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This part is an improvement of part BBa_K2088015 </https://parts.igem.org/Part:BBa_K2088015>, which consisted by the same copper ion detecting promoter and GFP as reporter. Comparing with BBa_K2088015, our improvement is using RFP to replace GFP as reporter.

Improvement： This design modification is based on three reasons and has significant advantage:

1) Comparing with GFP, RFP has a visible color which can be observed by naked eyes without UV excitation, therefore, in same condition, our new part can be used for qualitatively easily. Moreover, since it is not necessary to have a UV light source for qualitatively detecting, the prototype device could be smaller and cheaper.

2) RFP fluoresces red-orange when excited, therefore, in case of quantitative use, it will be same as GFP.

3) Usually, red color means “dangerous” and green color means “safe”. In the case of heavy metal pollution detection, the detecting result means more “dangerous” and “alert”, thus, using red color instead of green is in keeping with common sense.


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.Curve of REP fluorescence intensity and culture time under different copper concentrations
Fig.2.Photos of E. Coli DH5α after culturing for 10 hours under different copper concentrations.

As shown in Figure 1, with the extension of culture time, the fluorescence intensity of eRFP gradually increased which indicates that the expression level of eRFP in the medium gradually increased. In other words, the copper detection function of this working system was working normally. The concentration of copper ions in the waste water is close to 3ml/L. The detection results are similar to measurement result of ICP. Besides, as shown in Figure 2, as the copper concentration increased, the color turned redder. It is proved that the copper detection function of this working system was working as we expected.

This part have been improved by 2019 iGEM team ASTWS-China

This part is improved by 2019 iGEM team ASTWS-China. To solve the problems of spreading genetic-engineered strains in the environment, we designed the improved system which could lead the strains death when IPTG is added to the culture medium. We added a composite part consisted of a lacI regulated promoter (BBa_R0010) and a lysis gene (BBa_K117000) following the existed part. The improved part has been registered with number BBa_K3152008(https://parts.igem.org/Part:BBa_K3152008). More details can be seen on https://2019.igem.org/Team:ASTWS-China/Improve.


This part have been improved by 2019 iGEM team Worldshaper-Shanghai

This part is improved by 2019 iGEM team Worldshaper-Shanghai. To impove the problem of background leakage, the BBa_K3311008 was inserted between copA promoter and Riboj. The improved part has been registered with number BBa_K3311009 https://parts.igem.org/Part:BBa_K3311009. More details can be seen on https://2019.igem.org/Team:Worldshaper-Shanghai/Improve.