Difference between revisions of "Part:BBa K4288010"
 
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ArsA-GFP
 
ArsA-GFP
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== Characterization by Fujian_United ==
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== Profile ==
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== BBa_K4288010 ==
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Name: pro-ArsA amilGFP
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Name: ArsR-amilGFP
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Base Pairs: 1200 bp
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Base Pairs: 2564 bp
  
 
Origin: Escherichia coli
 
Origin: Escherichia coli
  
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Properties: a tool to monitor the arsenic
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Properties: Gene technology for protecting patented bacterial strains
 
== Usage and Biology ==
 
== Usage and Biology ==
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ArsD was designed to response to the various concentration of arsenic, and fused amilGFP to monitor the arsenic concentration.
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ArsA was designed to response to the various concentration of arsenic, and fused amilGFP to monitor the arsenic concentration.
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== BBa_K3991001 ==
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== BBa_K3991000 ==
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Name: ArsR
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Name: ArsA
  
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Base Pairs: 351 bp
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Base Pairs: 1749 bp
  
 
Origin: Escherichia coli
 
Origin: Escherichia coli
  
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Properties: regulatory protein
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Properties: arsenic metallochaperone
 
== Usage and Biology ==
 
== Usage and Biology ==
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BBa_K3991001 is a coding sequence of ArsR. ArsR is an As(III)-responsive transcriptional repressor which is capable to control its own expression. The repressive effect of ArsR is alleviated by arsenic, antimony, and bismuth, as well as arsenate.
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The ArsA protein is an arsenite-stimulated ATPase and complexed with ArsB protein. Its function is to transport the arsenic.  
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Bacteria developed a mechanism against the arsenic pervasiveness. Many bacteria processed three genes, arsRBC. Five gene ars operons have two additional genes, arsD and arsA, called arsRDABC. The additional genes ArsD and arsA derived from E.coli. The arsRDABC operon are more resistant to As due to the ArsA-ArsB complex that catalyzes ATP-driven As/Sb efflux.
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Bacteria developed a mechanism against the arsenic pervasiveness. Many bacteria processed three genes, arsRBC. Five gene ars operons have two additional genes, arsD and arsA, called arsRDABC. ArsR is an As(III)-responsive transcriptional repressor, additional genes ArsD and arsA derived from E.coli. The arsRDABC operon are more resistant to As due to the ArsA-ArsB complex that catalyzes ATP-driven As/Sb efflux.
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== Construct design ==
 
== Construct design ==
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In order to develop a real-time tool for detecting the arsenic binding, promotor ArsD was designed to response to the various concentration of arsenic, and fused amilGFP to monitor the arsenic concentration. This DNA fragment was inserted into the expression vector pET28a.
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In order to develop a real-time tool for detecting the arsenic binding, promotor ArsA was designed to response to the various concentration of arsenic, fused to amilGFP to monitor the arsenic concentration. This DNA fragment was inserted into the expression vector pET28a.
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== Proof of function ==
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== Experimental approach ==
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1.1 GFP intensity
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[[File:T--Fujian united--BBa K3991008-figure 1.jpg|500px|thumb|center|Figure 1. GFP intensity in different concentration of As.]]
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[[File:T--Fujian united--BBa K4288010-figure 1.jpg|500px|thumb|center|Figure 1. GFP intensity in different concentration of As.]]
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The result demonstrated the relationship between the florescence intensity and the arsenic concentration ranging from 10ug/L to 200ug/L. Compared to cultivation time of 1h or 0h, the green curve of cultivation time of 2h showed the significant increasing GFP intensity. however, the higher concentration of arsenic (100ug/L) might inhibit the bacteria growth, so the GFP intensity decreased. According the result, 50ug/L induced the maximum florescence expression under ArsA promoter.
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The figure 1 demonstrated certain level of positive association between the florescence intensity and the arsenic concentration ranging from 10ug/L to 200ug/L. We monitor the GFP intensity at 0h, 1h, 2h and 3h. The result showed that after cultivation time1h, the florescence intensity has no significant variation. However, after 2 hours, the trend of GFP intensity increased with increasing concentration of arsenic, then become stable. According to the result, 20ug/L As induced the maximum florescence expression for ArsD. Although we test the bacteria in 3h, the result is still similar to that in 2h, indicating cultivating for 2h and 20ug/L As is enough for testing GFP intensity
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Reference
  
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Reference
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1. Silver, S. and L.T. Phung, BACTERIAL HEAVY METAL RESISTANCE: New Surprises.[J] Annual Review of Microbiology, 1996. 50(1):753-789.
  
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1. Lin, Y.-F., J. Yang, and B.P. Rosen, ArsD: an As(III) metallochaperone for the ArsAB As(III)-translocating ATPase.[J] Journal of Bioenergetics and Biomembranes, 2007. 39(5):453-458.
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2. Lin, Y.-F., J. Yang, and B.P. Rosen, ArsD: an As(III) metallochaperone for the ArsAB As(III)-translocating ATPase.[J] Journal of Bioenergetics and Biomembranes, 2007. 39(5):453-458.
  
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2. Wu, J. and B.P. Rosen, The ArsR protein is a trans-acting regulatory protein.[J] Molecular Microbiology, 1991. 5(6):1331-1336.
 
  
  

Latest revision as of 14:21, 12 October 2022


ArsA-GFP

ArsA-GFP

Profile

Name: pro-ArsA amilGFP

Base Pairs: 2564 bp

Origin: Escherichia coli

Properties: Gene technology for protecting patented bacterial strains

Usage and Biology

ArsA was designed to response to the various concentration of arsenic, and fused amilGFP to monitor the arsenic concentration.

BBa_K3991000

Name: ArsA

Base Pairs: 1749 bp

Origin: Escherichia coli

Properties: arsenic metallochaperone

Usage and Biology

The ArsA protein is an arsenite-stimulated ATPase and complexed with ArsB protein. Its function is to transport the arsenic.

Bacteria developed a mechanism against the arsenic pervasiveness. Many bacteria processed three genes, arsRBC. Five gene ars operons have two additional genes, arsD and arsA, called arsRDABC. ArsR is an As(III)-responsive transcriptional repressor, additional genes ArsD and arsA derived from E.coli. The arsRDABC operon are more resistant to As due to the ArsA-ArsB complex that catalyzes ATP-driven As/Sb efflux.

Construct design

In order to develop a real-time tool for detecting the arsenic binding, promotor ArsA was designed to response to the various concentration of arsenic, fused to amilGFP to monitor the arsenic concentration. This DNA fragment was inserted into the expression vector pET28a.

Experimental approach

Figure 1. GFP intensity in different concentration of As.

The result demonstrated the relationship between the florescence intensity and the arsenic concentration ranging from 10ug/L to 200ug/L. Compared to cultivation time of 1h or 0h, the green curve of cultivation time of 2h showed the significant increasing GFP intensity. however, the higher concentration of arsenic (100ug/L) might inhibit the bacteria growth, so the GFP intensity decreased. According the result, 50ug/L induced the maximum florescence expression under ArsA promoter.

Reference

1. Silver, S. and L.T. Phung, BACTERIAL HEAVY METAL RESISTANCE: New Surprises.[J] Annual Review of Microbiology, 1996. 50(1):753-789.

2. Lin, Y.-F., J. Yang, and B.P. Rosen, ArsD: an As(III) metallochaperone for the ArsAB As(III)-translocating ATPase.[J] Journal of Bioenergetics and Biomembranes, 2007. 39(5):453-458.