- Circuit Design and Parts
1. Lead and Lead Toxicity1
Lead(Pb) is a heavy metal with a low melting point; it can be combined with other metals to form alloy (see the summary). Lead is a ubiquitous environmental toxin that can cause many acute and chronic circulatory, neurological, hematological, and immunological pathologies. Lead is one of ten chemicals of major public health concern.1
The prime targets to lead toxicity are the heme synthesis enzymes, thiol-containing antioxidants and enzymes (superoxide dismutase, catalase, glutathione peroxidase, glucose 6-phosphate dehydrogenase and antioxidant molecules like GSH). The low blood lead levels are sufficient to inhibit the activity of these enzymes and induce generation of reactive oxygen species and intensification oxidative stress. Oxidative stress plays important role in pathogenesis of lead-induced toxicity and pathogenesis of coupled disease. Because brain is most sensitive to lead exposure, lead influences the development of the nervous system and is particularly harmful to children.1
2. Exposure Source or Route2
It has three forms: (1) naturally occuring ores, including galena (lead sulfide), anglesite(lead sulfate), cerussite (lead carbonate), mimetite (lead chloroarsenate) and pyromorphite (lead chlorophosphate); (2) inorganic lead: most in old paint, soil, dust and consumer products. Most common forms are white lead(lead carbonate compound), yellow lead (lead chromate, lead monoxide), or red lead (lead tetraoxide); (3) organic lead: tetra-ethyl lead is the form of lead used in leaded gasoline. Organic forms of lead are extremely dangerous. They are absorbed through skin and are toxic brain and central nervous system.2
3. Lead resistence in Cupriavidus metallidurans CH34
Many bacteria have developed or acquired genetic systems for heavy metal resistance in the worsening environmental contamination of heavy mental toxin. Taking Cupriavidus metallidurans CH34 as an example, it contains over 7 genes encoding resistances to toxic heavy metals, located on one of the two endogenous megaplsmids, pMOL 28 and pMOL30.
Figure 1. The lead transport system in C. metallidurans CH34 (based on Hynninen et al., 2010, modified)3
Figure 1 shows that the lead transport system in C. metallidurans CH34 (based on Hynninen et al., 2010). Pb2+, entering the cell through the Zn2+ and Mn2+ transporters, is actively pumped out by PbrA to the periplasmic space. Pb2+ precipitated with phosphates (P, released by the PbrB protein.) PbrD is a putative intracellular Pb2+ binding protein (Borremans et al., 2001; Hynninen et al., 2009). OM, outer membrane; IM, inner membrane. 3
Figure 2. Regulation of pbr by PbrR 3
The operon for pbr efflux (pbr) is located within megaplsmid pMOL30. Seven genes of the pbr locus (pbrUTRABCD) are organized into two divergently oriented transcription segments: pbrUTR and pbrABCD. The two pbr promoters of these two segments are both regulated by the transcriptional regulator PbrR. PbrR is a member of MerR family. However, unlike MerR proteins of the other metals, PbrR has only been found on plasmids in a few bacteria 3
1. Global Alliance to Eliminate Lead Paint 4
The Global Alliance to Eliminate Lead Paint is a cooperative initiative jointly led by the World Health Organization and the United Nations Environment Programme. This allilance’s goal is to prevent children’s from exposure to lead in paints and also to reduce lead exposures in paint in working environment. The Alliance's broad objective is to eliminate the risks of lead in paints. The Alliance is an important means of contributing to implementation of paragraph 57 of the Plan of Implementation of the World Summit on Sustainable Development and to resolution II/4B of the Strategic Approach to International Chemicals Management (SAICM).
2. Taiwan Regulation on Lead in Chinese medicine
Our government’s regulation on the lead in Chinese medicine sets a general limit of Pb<=5.0ppm for most Chinese medicine with exceptions:
(1) 8 kinds: not subject to any limits on heavy metal containment
(2) 25 kinds: subject to the limit of total heavy metal containment(<=20ppm; <=30ppm), not specific for lead.
(3) 8 kinds: not subject to lead limit.
(4) 19 kinds: are subject to lead limit of (<=10ppm)
(5) 8 kinds: are subject to lead limit of (<=15ppm)
Circuit Design and Parts
1. Idea for Pb2+ Biosensor
The idea of Pb2+ biosensor is involved with two steps: (1) generation PbrR protein with a binding site for lead ion, forming a Pb2+-PbrR dimer (2) GFP expression when the Pb2+-PbrR dimer binds onto the pbr promoter downstream. Thus, the GFP expression could be observed in two occasions
(1). Pb2+ binds onto the PbrR which binds onto the Lead promoter first
Figure 3a. Lead biosensor Circuit 1
(2). Pb2+and PbrR form a Pb2+-PbrR dimer to bind onto the Lead promoter and activate
Figure 3b. Lead biosensor Circuit 2
From the literature we understand that PbrR and Pb2+-PbrR dimer compete for the binding sites for pbr promoters. When the amount of PbrR are larger than Pb2+, GFP expression will be affected.
Figure 3c. Lead biosensor Circuit 3
Thus, we established an idea to control the amount of PbrR generation by using the autoregulation system. In this way, we can precisely compare the amount of PbrR generation through GFP expression.
2. Circuit Design for PbrR generation
Thus, we design a circuit with autoregulation mechanism for our PbrR generation(BBa_K1961004), which is based on the control of a PTet promoter with specific binding sites for TetR in front of a GFP. The TetR is repressor. We use tetracycline to induce the generation of PbrR, GFP and TetR. The generated TetR can repress the generation of TetR and PbrR. Using this autoregulation, we can detect the production of PbrR through fluorescence analysis.
Figure 4. Autoregulated PbrR generator
We will compare flurescence expression autoregulation with another circuit of PbrR generator(BBa_K1961005), which is under general promoter.
Figure 5. Common PbrR generator
1. Simulation Result of Autoregulated PbrR generation
Figure 6. The Autoregulation circuit and the related rate equations
Figure 7. The parameter space for constitutive circuit
Figure 8. The fold analysis for autoregulation and constitutive circuits
2. Fluorescence of Lead Biosensor
This was a previously existing composite part(BBa_K1613017) can detect lead ions. In 2015, HSNU-TAIPEI didn’t succeed and didn’t ship this part. This year, we re-do the ligation of this composite part as new part (BBa_K1961006) and tested its fluorescence.
Figure 9 Fluorescence of Lead Biosensor in different concentrations of lead solution(Part BBa_K1961006)
Figure 10 Fluorescence/OD of Lead Biosensor in different concentrations of lead solution(Part BBa_K1961006)
 Lead poisoning. Wikipedia. https://en.wikipedia.org/wiki/Lead_poisoning
 WHO 2010. Childhood Lead Poisoning. WHO Press. http://www.who.int/ceh/publications/leadguidance.pdf
 Anna Jarosławiecka and Zofia Piotrowska-Seget. Lead resistance in micro-organisms. Microbiology (2014), 160, 12–25
 Global Alliance to Eliminate Lead Paint. http://www.who.int/ipcs/assessment/public_health/gaelp/en/