Contribution

Overview

Studies show that not only is heavy metal pollution a severe issue, it is also prevalent around the world, especially in water sources. Furthermore, water contamination may accumulate in soils and sediments. In China, around 16.1% of arable land is potentially contaminated by heavy metals.

Humans then take them in without notice, either by directly drinking contaminated water or through eating other plants and animals that are previously exposed to heavy metals. If the heavy metals enriched in human bodies, they can cause serious symptoms such as diseases in the kidney, the lung, the liver, the bone, different types of cancers, and deficiency in embryonic neurodevelopment.

Add new functional data to an existing part BBa_K3991007, pro-ArsR-amilGFP

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.

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.

Proof of function

p1.1 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

Add new functional data to an existing part BBa_K3991006, pro-ArsD-amilGFP

The ArsD homodimer functions as an arsenic metallochaperone which bound to As. The complex of ArsD-As interacts with ArsA to active its ATPase activity.

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 ArsD 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.

Proof of function

1.1 GFP intensity

Figure 2. GFP intensity in different concentration of As

The figure2 demonstrated the relationship between the florescence intensity and the arsenic concentration ranging from 10ug/L to 200ug/L. The higher concentration of arsenic (>100 ug/L) might inhibit the bacteria growth, so the GFP intensity decreased. At 2 hours, 50ug/L is the maximum florescence expression for ArsD, then become stable.

Add new functional data to an existing part BBa_K3991008, pro-ArsA-amilGFP

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 3. 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. 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.
2. Wu, J. and B.P. Rosen, The ArsR protein is a trans-acting regulatory protein.[J] Molecular Microbiology, 1991. 5(6):1331-1336.
3. Yang, J., S. Rawat, T.L. Stemmler, et al., Arsenic Binding and Transfer by the ArsD As(III) Metallochaperone.[J] Biochemistry, 2010. 49(17):3658-3666.
4. Silver, S. and L.T. Phung, BACTERIAL HEAVY METAL RESISTANCE: New Surprises.[J] Annual Review of Microbiology, 1996. 50(1):753-789.