Part:BBa_K3991000

ArsD

Profile

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.

Experimental approach

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

Figure 1: 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 2: Gel Electrophoresis Results of PCR of ArsD Gene..

Figures 2 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 3: 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 3 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

Fig4. 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]