Detecting

Summary

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. 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 defend metal ions including Zn²⁺, Co²⁺, Ni²⁺, Pb²⁺ and Cd^{2+ [1-3]}. Briefly, the protein SmtB generally functions 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. In previous reports, the smtB-OP-smtA element which located in Synechococcus may also function as a metal ions (Zn²⁺, Co²⁺ and Cd²⁺) responsive repressor in E. coli ^[4-6]. In this case, we focused on detecting the content of Cd²⁺, while whether this well-known element could be employed in our project still remained uncertain. Therefore, we took steps of experiments and yielded several interesting data below.

Firstly, we obtained the smtB-OP-smtA device from Synechococcus genome and tested the growth of bacterium containing this element in Cd²⁺ present condition (Figure 1). The result displayed a significant growth advantage with smtB-OP-smtA positive E. coli, indicating the device worked in E. coli. Thus, we wanted to use pigments as reporters in our designed genetic construct which can be recognized by the naked eye. According to the previous work, we have chosen an identified pigment in the Registry: the biobrick of RFP (BBa_E1010) 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).

It was important to us that the similar device which contained Lac-Z as reporter gene had ability to sense Zn²⁺ and Cd²⁺ in E. coli [7, 8]. Thus, there are four potential metal binding sites on protein SmtB as known as α3, α3N, α5 and α5C ^[9], different from Zn²⁺ resistance mechanisms, Cd²⁺ binds SmtB at the site α3N ^[10]. To gain deep insight regarding the detailed comparison of smtB-OP-reporter affected by these two metal ions in E. coli, we adopted Rosetta-plysS and detected the expression of reporter gene by measuring OD value (OD450 for RFP). As shown in Figure.2 A-B, pigment gene can be both activated by Zn²⁺ and Cd²⁺, the up-regulation from Cd²⁺ was with more sensitive with shorter period and lower consistency than Zn²⁺. With higher density of bacteria (1x10⁷ cells ml^-1), the pigment expression induced by Cd²⁺ in 2 hours could be easily distinguished by our naked eye (Fig.3C). Taken together, these data provided basic but necessary information that we could take this recombinant device to report the content of Cd²⁺ with certain degree (2-20μM in 2 hours) not affected by Zn²⁺.

Based on the conclusions above, we focused on the details of our detecting device resulting from Cd²⁺ only with certain degree. For quality control, we detected the standard substances by Optima 8300 ICP-OES Spectrometer the credible interval of this method displayed to land at 50-500μM (Table.1). For the pigment expression differences with Cd²⁺ at 10-50μM in 2 hours are not obvious, our team has coupled a more impressible biobrick part amilCP (BBa_K592009) with the smtB-OP element. Results from Fig 3 A-B indicated that, the diversity of amilCP expression (measured by OD600) was able to represent the continuous concentrations of Cd²⁺ precisely within 0-50μM in one hour. The visualization is another potential advantage of this system, we raised the dose of the engineering bacteria and extended the sense time. Finally, blue color produced by our device was strong enough to be observed by the human naked eye, however, similar lightness among 100-500μM Cd²⁺-treatment samples were shown which matched the previous data (Fig.3 C).

In summary, the Rosetta-plysS strain made our system convenient to be applied, the classical smtB-OP-smtA device from Synechococcus supported our system a responsive Cd²⁺ inducible-promoter, and the viewable pigment gene provided our system a reliable and macroscopic observation. After theoretical prediction, genetic engineering, experimental optimization and reasonable model analysis (deeply discussed in Modeling), our detecting system residing in the engineering bacteria was able to sensitively represent the content of Cd²⁺ (1-100μM) in 1-2 hours.

Although the inducible operator in our case might also response to other metal ions including Zn²⁺, our date at least did point out that Cd²⁺ has acuter stimulus to the pigment gene than Zn²⁺ which was confirmed both from experimental data and model analysis. We achieved our goal at a certain degree. Finally, our system is easier to utilize and exhibits improved flexibility as a tool to detect Cd²⁺ which belongs to the toxical heavy metal ions.

Protocols

Plasmid design and construction

The vector pHY300 PLK and vector PACYC184 were constructed based on the smtBCP/smtBRP backbone (see obtaining the target genes) using standard cloning techniques. The sensor vector included an improved metal ions inducible promoter (smtO-P), a pigment gene (RFP or amilCP) marker. Detailed vector maps, sequence information and cloning protocols has been described above in Foundation.

Establishment of the detecting clonal cell lines.

DNA transfection was performed as standard molecular cloning techniques, the Rosetta-plysS strain (kindly supported by our instructors) was cultured with LB medium. All the clonal cells were validated by PCR (see Foundation).

OD value measurement and analysis.

All measurement procedures were performed using an Eppendorf BioSpectrometer basic instrument. Briefly, the cells were centrifuged at 3,000g for 5 min. Then, resulting pellet was twice-washed with ddH₂O and resuspended at a density of 10^5-6 cells ml^-1. After that, the cells were treated with metal ions using a shaker culture box with 200rpm at 37^。C. All the data were analyzed using Sigmaplot software. For each experiment, triplicate cultures were measured.

Quality control of the Cd²⁺ standard samples by emission spectrometric detection.

Briefly, the CdCl₂ (Sigma-Aldrich) was taken for preparing the standard samples. 183.32g (1M) was weighed by analytical balance and diluted at a concentration of 1M with ddddH₂O. After 10-times-dilution step by step, we got several standard samples of Cd²⁺ at different concentrations (1μM, 10μM, 50μM, 100μM and 500μM). Then, these samples were measured via Optima 8300 ICP-OES Spectrometer (this section was performed in HIT). Detailed condition for the detection was shown in table.2. For each experiment, 8 separate repeats were measured.

Figure and Table

Figure 1

Fig.1. Growth of cells containing smtB-OP-smtA element in LB medium supplemented with 2μM Cd²⁺. Cells were inoculated at a density of 1x10⁶ cells ml^-1, and growth was monitored by measuring the OD₅₄₀ value. Data points represent the mean values from three separate cultures with SD.

Figure 2

Fig.2. A. Metal-induced expression of the pigments (RFP), Rosetta-plysS (1x10⁶ cells ml-1) carrying the smtB-OP-RFP element were grown with Cd²⁺ and Zn²⁺ (1-50μM) supplement for 2h immediately before assay and expression was monitored by measuring the OD₄₅₀ value; B. Metal-induced expression of the pigments (RFP). Rosetta-plysS carrying the smtB-OP-RFP element were grown with Cd²⁺ and Zn²⁺ (2μM) supplement for 1-12h immediately before measurement; C. Cadmium-induced expression of the pigment (RFP) at different concentrations. Rosetta-plysS (1x10⁷ cells ml^-1) carrying the smtB-OP-RFP element were grown with Cd²⁺ (1-20μM) supplement for 2h. The data points shown in A and B represent the means of three separate assays with SD.

Table 1

Expected concentration(μM)	1	10	50	100	500
Reported measured(μM)	0.85±0.19	9.68±1.28	55.57±5.66	107.52±11.98	528.07±33.15

Figure 3

Fig.3. A. Cadmium-induced expression of the pigment (amilCP) at constant concentration. Rosetta-plysS (1x10⁶ cells ml^-1) carrying the smtB-OP-amilCP element were grown with Cd²⁺ (1μM) supplement for 1-2h immediately before assay and the expression was monitored by measuring the OD₆₀₀ value; B. Cadmium-induced expression of the pigment (amilCP) with different concentrations. Rosetta-plysS carrying the smtB-OP-amilCP device were grown with Cd²⁺ (1-100μM) supplement for 1h immediately before assay;C. Cadmium-induced expression of the pigment (amilCP) with different concentrations. Rosetta-plysS (1x10⁷ cells ml^-1) carrying the smtB-OP-amilCP element were grown with Cd²⁺ (10, 20, 50, 100, 200 and 500 μM) supplement for 2h. The data points shown in A and B represent the means of three separate values with SD.

Table 2

RF power	1.3kw
flow rate of plasma	14L/min
flow rate of assistant gas	0.2 L/min
flow rate of atomization	0.55 L/min
the flow velocity of peristaltic pump	1.5 L/min
the time of washing the sample	40 sec
integration time	5 sec
method	axial observation
the wavelength of Cd2+	228.802 nm

Reference

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