Part:BBa_K3991008

pro-ArsA-amilGFP

We have made new contribution based on the past.

BBa_K3991008

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.

The previous infos are as follows:

Usage and Biology

According to the WHO, arsenic levels above 10 parts per billion in water are harmful to humans. Levels in Bangladesh, however, are five times as much. The Bangladesh arsenic contamination is pending a solution. "I have no alternative." Uddin, a villager in Bangladesh, helplessly expresses his views towards drinking arsenic-contaminated well water.

What comes to your mind first when talking about arsenic? Arsenic is a naturally occurring element that is widely distributed in the earth's crust. It is found in water, air, food, and soil. In recent years, reports of arsenic poisoning have been increasing year by year.

Arsenic seeps into groundwater through rocks and soil, resulting in drinking water from surface sources (such as wells) that often contain higher levels of arsenic than water from, for example, lakes or reservoirs. In addition to groundwater, arsenic levels of 1.7 μg/L have been detected in the ocean, far exceeding the international regulation of 0.0175 μg/L (Neff, 2009). Irrigation with arsenic-contaminated water sources makes arsenic hazardous to human health from the food side. In addition, arsenic can also enter the human body from external sources, such as paints, textiles, and metal adhesives, through direct ingestion, gaseous inhalation, or skin absorptio. Or it can be absorbed by humans as a component of tobacco (WHO, 2018). Long-term exposure to high levels of arsenic can be harmful to humans, especially to developing infants and children. Although arsenic is not well understood, once it enters the body, the skin and various systems such as the nervous, respiratory, cardiopulmonary, immune, and endocrine systems are affected. In addition, the liver, kidneys, bladder, and prostate, which are responsible for detoxification, are damaged and cannot function effectively (National Institute of Environment Health Science, 2021).

For example, in a village in Hunan, China, 1200 in total 3000 residents have been tested for arsenic poisoning, which was mainly caused by the mining of realgar ore. According to the local hospital, 400 of 600 miners who have been tested for arsenic poisoning died from cancer. For instance, a family of 7 people all died from cancer, and 5 of the cases were determined that they were caused by arsenic poisoning (Chinese Center for Disease Control and Prevention, 2014).

Not only for humans but excessive levels of arsenic can also affect plants and animals in the natural environment. For example, aquatic plants (e.g. algae), zooplankton, and amphibians, or aquatic animals (e.g. snails, fish, crustacean larvae, marine mammals) are all exposed to inorganic arsenic toxicity (Neff, 2009). Arsenic is a microelement that is omnipresent in the environment. However, it is this common microelement that could be harmful to human body. If we take in more than 50 µg/L of arsenic, the function of our body will be disrupted. Also, the level of arsenic throughout the world is gradually increasing every year, and has already exceeded the level that human body can metabolize. Areas close to factories whose product involves arsenic usually have a high arsenic concentration. Thus, we need arsenic detectors to ensure the security of people who work in these factories. According to the questionnaires and street research we did, although the general public did not know much about arsenic, most people believed that having an inexpensive and accurate sensor to detect arsenic is necessary. In addition, environmental and market scientists also considered that the current testing methods in the market are too cumbersome, so equipment that can detect arsenic levels easily and efficiently is required.

Construct design

Figure 1. Plasmid diagram..

BBa_K3991002

Properties: Gene technology for protecting patented bacterial strains

ArsA ATPase is the catalytic subunit of the pump protein, which combines the hydrolysis of ATP with the movement of arsenic and antimony through the transmembrane ArsB protein. The organism Escherichia coli excretes arsenic from the body through this gene pathway, thereby reducing the poisonous effect of arsenic.

BBa_K592010

Usage and Biology

This part is useful as a reporter and it naturally exhibits strong yellow color when expressed.

In the process of cultivating microorganisms, namely Escherichia coli, because of their small size, they cannot be directly observed with the naked eye, and a certain method is needed for monitoring. In cell biology and molecular biology, the green fluorescent protein (GFP) gene is often used as a reporter gene. Through genetic engineering technology, the green fluorescent protein (GFP) gene can be transferred into the genomes of different species and continue to be expressed in offspring. . Therefore, we constructed the amilGFP engineered bacteria to show the growth status (number and vitality) of E. coli by observing the strength of the green fluorescent protein signal.

Experimental approach

Production, purification, and sequcing analysis of recombinant ArsA-amilGFP

Figure 2: Gel Electrophoresis Results of PCR of amilGFP Genes..

Figure 1 shows gel electrophoresis results of amilGFP PCR. Column M is a 2K marker ladder. Columns 1-6 are PCR products of amilGFP genes.

All 1-6 columns displayed successful results at 700bp which could be used for later experiments. 

Figure 3: Gel Electrophoresis Results 1 of Colony PCR of ArsA..

Figures3 show the result for colony PCR identification on the E.coli with ArsA inserted that were cultivated previously. The purpose is to examine whether the E.coli contains expected gene segment of ARSA.

..

References

(15)Arsenic W.H.O. World Health Organization. February. 2018. [Accessed August 3, 2018]. Available from: http://www.who.int/news-room/fact-sheets/detail/arsenic.

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 1590
  
• 1000
  COMPATIBLE WITH RFC[1000]