Project

Overview

In recent years, a series of cases of cadmium pollution in our country have triggered wide concerns among Chinese people. Irrigating water was contaminated by cadmium raw sewage discharged from industry, then it caused the high levels of cadmium in farmland, while the rainfall worsened this process by releasing cadmium trapped in soil polluted by industrial solid waste. Considering the properties of heavy metals, unable to decay and thus a different kind of challenge for medication, we have constructed this bacteria designated as Nanocrystal E. coli Flocculation Units. The designed bacteria in our project consists of three modules in function. The first function is a detecting system that displays the presence of cadmium ions. The second function is a recycling system that synthesizes the cadmium sulfide nanocrystals to recycle ions. The last function is a flocculating system to increase the flocculent activity of the bacteria containing cadmium sulfide nanocrystals so that we can easily collect the nanocrystals formed by our engineered bacteria, as well as to avoid secondary pollution. Eventually, we can realize our double-win goal, safeguarding our environment by removing heavy metal ions and yielding available nanocrystals.

Background

Cadmium, with symbol Cd, is a soft bluish-white metal. It was discovered in 1817 simultaneously by Stromeyer and Hermann. The average concentration of Cd in the Earth's crust is between 0.1 and 0.5 parts per million (ppm).

The element is mainly used in Ni-Cd batteries and protective coating and enters the environment through both natural and numerous anthropogenic sources [1]. Currently, about three-quarters of Cd usage goes into the production of batteries and most of the remaining quarter is used for pigments, corrosion resistant coatings and plating, and as a stabilizer for plastics. During the 20th century, the production of Cd increased tremendously, especially after World War II. Although it went through some ups and downs, the overall trend is still increasing.

Fig.1. World cadmium output during the last 100 years.

Only limited amounts of Cd in products are recycled. Cd in consumer products primarily ends up at dump sites or incinerators, exposed to the environment (Fig2, Fig3). As it’s exposed to the environment, it has been very close to living organisms. Leafy plants such as tobacco, rice, and lettuce can soak up Cd as if it were any other soil nutrient. Through tobacco smoking, smokers have 4-5 times higher blood Cd concentrations and 2-3 times higher kidney Cd concentrations than non-smokers. Besides tobacco smoking, diet is the leading source of Cd exposure for the non-smoking general population. The rice absorbed the Cd, and then the Cd accumulated in the people eating contaminated rice. Unfortunately, in Japan, Korea, Taiwan, China and other countries of Asia, rice is the major source of diet. It means the main route of environmental Cd exposure for the general population is through the consumption of contaminated food.

Fig.2. Contaminated soil

Fig.3. Cd pollution degrees in China

With other nutritional stress like Fe, Zn, or Ca deficiencies, high dietary intake of Cd can leads to associated human Cd diseases. As a non-essential element for human bodies, Cd shares the same period in the periodic table with Zn, which is an essential metal for human bodies. They share a common oxidation state (bivalent) and are almost the same size when ionized. Due to these similarities, Cd can replace Zn in many biological systems, in particular, system that contains softer ligands such as sulfur. But Cd forms bonds up to ten times stronger than Zn in certain biological systems, so it’s notoriously difficult to remove from other compounds. The estimated half-life of Cd in the human system exceeds 15 years. The prolonged intake of even small amounts of Cd leads to skeletal damage and dysfunction of the kidneys. That makes them toxic in mammals.

Most researchers took the Itai-Itai disease (Fig4) as a favorite example when discussing about Cd contamination and human Cd diseases, for it’s the first documented case of mass Cd poisoning in the world. The name coined by locals describes the screams due to the severe pain in bones. It’s still one of four major pollution-related diseases in Japan now.

Fig.4. Itai-Itai disease

Although it’s outbreak was in around 1912, we are still plagued by the same incidents constantly. Many paddy rice-growing areas in our country have been reported containing high levels of Cd heavy metal. As much as 10 percent of China's rice may be tainted by poisonous Cd. Local farmers are aware that eating the tainted rice they grow can make them sick, but they can't afford the cleaner rice sold in markets. Many other farm families have been kept in the dark about the heavy metal hazards due to a lack of information about pollution risks, so they continue eating toxic rice. To date, none of China's hospitals have officially recognized the weak leg condition common in some regions. But agriculture scientists have reluctantly called the phenomenon an early symptom of Itai-Itai disease.

As you can see above, Cd is widely used, hard to decay, thus little has been recycled. A considerable amount of Cd has become an uninvited guest in the chain of food, then leave us the chaos of human Cd diseases. So we decided to do something about it.

Reference

1. Besante, J., J. Niforatos, and A. Mousavi, Cadmium in Rice: Disease and Social Considerations. Environmental Forensics, 2011. 12(2): p. 121-123.

Design

The enginerring bacteria designated as Nanocrystal Flocculation E. coli Units seems feasible for our purposes. Each of our unit consists of three functional modules. The first one is a detecting system that displays the presence of cadmium ions. The second one is a recycling system that is able to synthesize the cadmium sulfide nanocrystals to recycle ions. The last one is a flocculating system to increase the flocculent activity of the bacteria containing cadmium sulfide nanocrystals to easily collect the nanocrystals formed by the bacteria, as well as to avoid secondary pollution. In addition to detecting and depleting cadmium from water, we intended to make a fundamental change to convert this toxic contaminant to a popular semiconductor-nanocrystal, which can be applied to devices for photoelectronics and fluorescent probes in biological system.

Principles

As we know, some metal ions are toxic to bacterial cells at all concentrations, therefore detoxification and resistance systems that employ a variety of mechanisms to rid the cell of these potentially lethal toxins have evolved employ. In most cases, the expression of such resistance systems is controlled at the level of transcription by metal sensor proteins that sense specific metal ions via their direct coordination [1]. The most famous resistance system is smtB-OP-smtA device which defends metal ions including Zn²⁺, Co²⁺, Ni²⁺, Pb²⁺ and Cd²⁺[2-4].

Briefly, the protein SmtB which has four potential metal binding sites as known as α3, α3N, α5 and α5C [4] generally function as a repressor in the absence of metal ions and become activators upon metal binding, by driving a metal-induced DNA conformational switch that converts a sub-optimal promoter (OP-promoter) into a potent one, then activate the expression of smtA. And different from Zn²⁺ resistance mechanisms, Cd²⁺ binds SmtB at the site α3N [5].

In previous reports, the smtB-OP-smtA element which located in Staphylococcus may also functions as a metal ions (Zn²⁺, Co²⁺ and Cd²⁺) responsive repressor in E. coli [6-8].

In our project, we wanted to use pigments as reporters in our designed genetic construction which can be recognizable by the naked eyes. According to the previous work, we have chosen an identified pigment in the Registry: the biobrick of amilCP (BBa_K592009) was used as reporter gene in our metal detection device. After exchanging the biobrick part with SmtA in smtB-OP-smtA device, the pigment gene was under control of metal-induced promoter (smtB-OP).

Reference

1. Busenlehner LS, Pennella MA, Giedroc DP. The SmtB/ArsR family of metalloregulatory transcriptional repressors: Structural insights into prokaryotic metal resistance. FEMS microbiology reviews. 2003;27(2-3):131-43.
2. Robinson NJ, Whitehall SK, Cavet JS. Microbial metallothioneins. Advances in microbial physiology. 2001;44:183-213.
3. Gutierrez JC, Amaro F, Diaz S, de Francisco P, Cubas LL, Martin-Gonzalez A. Ciliate metallothioneins: unique microbial eukaryotic heavy-metal-binder molecules. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry. 2011;16(7):1025-34.
4. Cook WJ, Kar SR, Taylor KB, Hall LM. Crystal structure of the cyanobacterial metallothionein repressor SmtB: a model for metalloregulatory proteins. Journal of molecular biology. 1998;275(2):337-46.
5. Busenlehner LS, Cosper NJ, Scott RA, Rosen BP, Wong MD, Giedroc DP. Spectroscopic properties of the metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC. Biochemistry. 2001;40(14):4426-36.
6. VanZile ML, Chen X, Giedroc DP. Allosteric negative regulation of smt O/P binding of the zinc sensor, SmtB, by metal ions: a coupled equilibrium analysis. Biochemistry. 2002;41(31):9776-86.
7. Morby AP, Turner JS, Huckle JW, Robinson NJ. SmtB is a metal-dependent repressor of the cyanobacterial metallothionein gene smtA: identification of a Zn inhibited DNA-protein complex. Nucleic acids research. 1993;21(4):921-5.
8. Huckle JW, Morby AP, Turner JS, Robinson NJ. Isolation of a prokaryotic metallothionein locus and analysis of transcriptional control by trace metal ions. Molecular microbiology. 1993;7(2):177-87.

Plasmids

Abbreviations
B	smtB	Trans-acting regulator
OP	smtO-P	Smt operator/promoter region, a bi-directional promoter
A	smtA	Encoding MT-like protein that can sequester metal ions
C	amilCP	Encoding a chromoprotein that has a blue/purple color visible to the naked eyes. A registered part from iGEM11_Uppsala-Sweden
R	RFP	Red Fluorescent Protein. A registered part from iGEM11_Uppsala-Sweden
Flo	Flocculation gene	It can improve the flocculent activity of our host cells (Rosetta pLysS)
CP25		A strong constitutive promoter
CDS7		Encoding a short peptide that can bind to CdS and induce the formation of CdS nanocrystals
BCP		According to priority: smtB, smtO-P(omit here), amilCP
BRP		According to priority: smtB, smtO-P(omit here), RFP
OPA		According to priority: smtO-P, smtA
FCDS7		According to priority: flocculation gene, CP25(omit here), CDS7

Totally, 5 plasmids will be constructed:

1. pHY300PLK-BCP-OPA
2. pHY300PLK-BRP-OPA
3. PACYC184-BCP-OPA
4. PACYC184-BRP-OPA
5. pET-28b(+)-FCDS7

As you can find above, in order to achieve the two goals of (a) detection by reporting the concentration of heavy metal cadmium and (b) forming CdS nanocrystals followed by flocculating when needed, we need 2 plasmids with their one replication origin different from another and different antibiotics so that they can adapt themselves and replicate in one host cell. To make sure that the backbone fits the host cell and the system we design, we chose 2 types of vectors with different copy numbers to see which one is fitter. And to make sure the reporter module can work well, we chose 2 reporter genes from previous registered parts (iGEM11_Uppsala-Sweden), amilCP (BBa_K592009) and RFP (BBa_E1010) to see which one is better. So that adds up to 4 plasmids for function (a). As for function (b), the vector we selected is pET-28b(+).

FIRST plasmid designation:

Gene smtB and smtO-P will be spliced with the reporter gene RFP by SOE PCR. And the product as a new target one (RCP) will be inserted into the backbone by double digestion with the chosen enzymes BamH I & EcoR I and ligation with ligation kit. In order to maintain the function of the original smt locus we will insert smtO-P and smtA (OPA) into the backbone at another site also by double digestion with the chosen enzymes BssH II & Nsi I and ligation with ligation kit.

SECOND plasmid designation:

The same principle as the FIRST, change the reporter gene into amilCP, splice it with partial smt locus by SOE PCR, insert the product (BRP) by double digestion(BamH I & EcoR I) and ligation, insert another partial smt locus by double digestion(BssH II & Nsi I) and ligation.

THIRD plasmid designation:

PACYC184 backbone has lower copy number than pHY300PLK. We presume it’s more suitable for the expression. BCP will be inserted by single enzyme digestion with Sac II and ligation with ligation kit. Meanwhile OPA will be inserted by using the Xba I site.

FOURTH plasmid designation:

Similarly, BRP will be inserted by single enzyme digestion with Sac II and ligation with ligation kit. Meanwhile OPA will be inserted by using the Xba I site.

FIFTH plasmid designation:

This plasmid is designed for function (b). In order to combine the detection and recycling, we wish the reconstructed bacteria could form CdS nanocrystal constantly at the presence of Cd²⁺ and S^2-. So we added a strong constitutive promoter CP25 in front of CDS7, whose product is a short peptide that can bind to CdS and induce the formation of nanocrystal as well as make it more biocompatible. And the upstream of the constitutive promoter is the flocculation gene, which is expected to be responsible for the flocculation of the bacteria, and it is under the control of T7 promoter, which is already on the selected vector.

Future Application

Via successful application of module 1 and module 2 above, we achieved the goal that our engineering bacteria displayed the presence of Cd2+ and synthetized nanocrystals. However, there is another problem coming, the 2nd pollution committed by bacterium themselves spreading limits its future application and conceals the function of module 2. In our opinion, the rapid flocculation of our engineering bacteria is viable in avoiding this bottleneck and helpful in collecting the solid contaminant. As reported, flocculating systems are capable to increase the flocculent activity of the bacteria, a flocculation gene was tentatively adopted in our next work. In this section, our host bacteria containing a flocculation gene cloned from Bacillus sp. F2 was presented and detected. In total, this work supported our project with convenience in enriching the nanocrystals and reliability against secondary pollution. Eventually, we can realize our double-win goal, safeguarding our environment by removing heavy metal ions and yielding available nanocrystals. However, this work was limited in the laboratory. See future application in Results.