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ArsA-GFP

Characterization by Fujian_United

BBa_K4288010

Name: ArsR-amilGFP

Base Pairs: 1200 bp

Origin: Escherichia coli

Properties: a tool to monitor the arsenic

Usage and Biology

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

BBa_K3991001

Name: ArsR

Base Pairs: 351 bp

Origin: Escherichia coli

Properties: regulatory protein

Usage and Biology

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. 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.

Construct design

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.

Proof of function

1.1 GFP intensity

Figure 1. GFP intensity in different concentration of As.

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

Reference

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.

1. Wu, J. and B.P. Rosen, The ArsR protein is a trans-acting regulatory protein.[J] Molecular Microbiology, 1991. 5(6):1331-1336.