Part:BBa_K3991006

pro-ArsD-amilGFP

We have made new contribution based on the past.

BBa_K3991006

Name: pro-ArsD-amilGFP

Base Pairs: 1200 bp

Origin: Escherichia coli

Properties: Gene technology for protecting bacterial strains

Usage and Biology

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

BBa_K3991000

Name: ArsD

Base Pairs: 1200 bp

Origin: Escherichia coli

Properties: arsenic metallochaperone

Usage and Biology

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 1. GFP intensity in different concentration of As.

The figure1 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.

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. 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.

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_K3991000

Properties: Gene technology for protecting patented bacterial strains

ArsD is a trans-acting repressor of the arsRDABC operon that confers resistance to arsenicals and antimonials in Escherichia coli.

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 ArsD-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 of PCR of ArsD Gene..

Figures 3 show the result for colony PCR identification on the E.coli with ARSD/amilGFP inserted that were cultivated previously. The purpose is to examine whether the E.coli contains expected gene segment of ARSD.

Function Tests

Function Test With NaH2PO4, Attempt 1

Such function tests were performed under the hypothesis that NaH2PO4 is structurally similar to arsenic compounds that were meant to be detected. NaH2PO4 is used to substitute for arsenic compounds due to experimental safety. No fluorescence was detected in all samples, indicating that the concentrations of NaH2PO4 may be too low or that genetically engineered E. coli will not react to NaH2PO4.

Function Test With NaH2PO4, Attempt 2

In order to verify our assumption in attempt 1, NaH2PO4 was used again in this testing, with greater concentration than the last test. No fluorescence was detected in all samples, indicating the inability of the genetically engineered E. coli to react with NaH2PO4.

Function Test With C2H6AsNaO2

Figure 4: Fluorescence Intensity of Transformed E. coli In Different Concentrations of C2H12AsNaO5 Solutions..

Given that NaH2PO4 cannot be used to stimulate the expression of amilGFP in genetically engineered E. coli, C2H6AsNaO5 was used for this function test. Extremely minor fluorescence was detected by a microplate reader after 19 hours of reaction time. This result confirms the design of E. coli to be correct and functional.

Figure 4 displays the fluorescence intensity generated by an ARSD/amilGFP transformed E. coli reacting for 1 hour in C2H6AsNaO5 solutions. As seen in the figure, fluorescence was detected for all C2H6AsNaO5 concentrations that are above 0, which confirms that the designed plasmid worked as intended. However the fluorescence was rather minor, so we speculate that E. coli responds poorly to organic arsenic compounds. Further experiments would be conducted to test for fluorescence intensities of E. coli in inorganic arsenic solutions.

Improvement of an existing part

Fig5. The blast results about the DNA sequence of our new part BBa_K3991006 and the old one BBa_K592010..

In order to optimize the function of BBa_K592010, we constructed an improved composite part BBa_K3991006, which is involved in the response of E. coli to heavy metal arsenic. The purpose of our product is to reduce individuals’ exposure to arsenic and prevent the harmful impact it might bring through accurately detecting the arsenic in people’s surroundings. After the successful construction of the engineering bacteria, on the one hand, it can help environmental monitors detect the pollution of heavy metal arsenic in the environment, and on the other hand, it can allow residents to detect whether there is heavy metal arsenic pollution in domestic water by themselves, thereby providing help for people's healthy life.

The improved strain can not only reflect the pollution situation according to the GFP signal, but also can be used for further transformation to reduce the pollution of heavy metal arsenic, such as promoting the enrichment of arsenic and then processing.

Future plan

Our main target customer are companies, factories, and government departments that being involved with arsenic detection. The main reason is that this detecting equipment can detect arsenic in a more efficient, convenient, and most importantly, more economical way. It is true to say that, the only conceivable drawback might be the accuracy of the result. However, the result is comparatively accurate enough to support our target clients to make basic decisions. Besides, we also take into account that some families with high pursuit of life quality, who worry about arsenic’s harmfulness and might become our potential customers, because these groups are usually educated to know the necessity of protecting their health, while purchasing specialized equipment is also affordable for them.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]