A lead-specific binding protein, PbrR, and promoter pbr from the lead resistance operon, pbr, of Cupriavidus metallidurans CH34 have shown highly sensitive and selective whole-cell detection of lead ions. The display of PbrR on the E. coli cell surface permitted selective adsorption of lead ions from solution containing various heavy metal ions. It is also one of our parts. We obtained it by DNA synthesis.
The following figure shows the genetic organization of the pbr operon locus in the C. metallidurans CH34 genome as well as some adsorption parameters of PbrR.
Fig 1. Genetic organization of the pbr operon locus in the C. metallidurans CH34 genome.
Fig 2. Selective adsorption of heavy metal ions by E. coli surface-displayed PbrR protein.
Fig 3. (a)−(c) The gradually magnified TEM images of PbrR protein surface-displayed E. coli cells after lead ion adsorption with the magnification from 20 000 to 100 000. (d), (e) The control TEM images of two randomly selected PbrR protein surface-displayed E. coli cells without lead treatment. (f) EDXA measurement of the red box from (c) representing the adsorbed lead ions.
GolB is a gold-binding chaperon discovered in S. typhimurium which is less than seventy amino acids in length. It has been verified that the display of GolB on E.coli cell surface permitted selective enrichment of gold ions from media containing various metal ions. It is one of our parts which is obtained by DNA synthesis by us.
The following figure shows the genetic organization of the gol locus in the S. typhimurium LT2 genome as well as the adsorption parameters of it.
Fig 4.Genetic organization of the gol locus in the S. typhimurium LT2 genome.
Fig 5. ICP-AES analysis of metal ions selective adsorption and recovery by E. coli surface-displayed GolB protein. The E. coli bacteria containing the OmpA–GolB plasmid with (+) and without (-) induction were grown in LB medium containing gradient concentrations of metal ions mixture.
Uranyl (UO22+), the predominant aerobic form of uranium, is present in the ocean at a concentration of ∼3.2 parts per 109 ; however, the successful enrichment of uranyl from this vast resource has been limited by the high concentrations of metal ions of similar size and charge, which makes it difficult to design a binding motif that is selective for uranyl. It has been reported that the design and rational development of a uranyl-binding protein using a computational screening process in the initial search for potential uranyl binding sites. The engineered protein is thermally stable and offers very high affinity and selectivity for uranyl with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity over other metal ions. Scientists also demonstrated that the uranyl-binding protein can repeatedly sequester 30–60% of the uranyl in synthetic sea water.
The picture below shows the crystal structure of the designed uranyl-binding protein SUP.
Fig 6 a). Competition assay of SUP versus total carbonate for uranyl yields a Kd of 7.4 fM at pH 8.9. The final solution of each point contained 10 mM protein, 10mM UO2 2+ and different concentrations of carbonate. b) Binding selectivity of SUP for uranyl over various other metals relevant to sea water extraction. Hatched columns, molar excess of ions in sea water; filled columns, selectivity of metal ions to uranyl by competition assay.
The Gram positive bacterium Bacillus subtilis has been extensively studied as a model prokaryotic system with which to understand gene regulation and the transcriptional control of unicellular differentiation. This organism is regarded as a non-pathogen and is classified as a novel food which is currently being used as a probiotic for both human and animal consumption. The distinguishing feature of this micro-organism is that it produces an endospore as part of its developmental life cycle when starved of nutrients. The mature spore, when released from its mother cell can survive in a metabolically dormant form indefinitely. The spore offers unique resistance properties and can survive extremes of temperature, dessication and exposure to solvents and other noxious chemicals. The protein component of the spore coat, such as Cot B, Cot C, Cot F, can be used as a fusion partner to express proteins or polypeptides on the spore surface, which we will further illustrate in the cell surface-display system.
Biofilms are assemblages embedded in a matrix composed of exopolysaccrides(EPSs), proteins and sometimes DNA. Matrix production results in the formation of complex architecture of typical of biofilms. In B. subtilis the extracellular matrix is composed of two major components, an EPS and the protein TasA, which polymerizes into amyloid-like fibers. Expression of the epsA-O operon, encoding the enzymes involved in EPS production, and tasA is indirectly under the control of the master transcriptional regulator Spo0A. Spo0A activity depends on its phosphorylation state. Spo0A phosphorylation is controlled by five Histidine kinases (KinA–KinE), which respond to different environmental cues. Phosphorylated Spo0A (Spo0A∼P) accumulation leads to the production of SinI, an antagonist of SinR. SinR is a transcriptional repressor that keeps the matrix genes shut off when conditions are not propitious for biofilm growth. When environmental cues that induce biofilm formation are present, the kinases are activated, and transcription of the matrix genes is induced via this signal transduction pathway.
It has been demonstrated that plant polysaccharides can stimulate biofilm formation in B. subtilis. It mainly functions in two ways: (i) by inducing matrix gene expression and (ii) by acting as a substrate that is processed and incorporated into biofilm EPS matrix.
Fig 7. Branda SS et al. Biofilms: The matrix revisited. Trends Microbiol 2005 13(1):20-26
In order to improve the efficiency of heavy metal adsorption, we decide to use the cell surface-display strategy which is especially advantageous over the traditional bio-adsorption methods because of the following considerations: First, the strategy alleviates the burden of intracellular accumulation of toxic metal ions, which often results in less adsorption efficiency and poor growth of the host cells. Second, the strategy allows for faster interaction between the cell surface-displayed metalloproteins and metal ions in the environment.
In B. subtilis, it is possible to use at least one other spore coat component, CotC， a small 8.8 kDa polypeptide, to display heterologous antigens, TTFC (51.8 kDa) and LTB (12 kDa). TTFC is non-toxic and immunogenic and expression in E.coli, yeast, Salmonella and Lactococcus lactis has been shown to provide protection against tetanus toxin challenge. LTB is the B subunit of the heat-labile toxin produced in enterotoxigenic strains of E. coli. The malleability and functional redundancy of both CotC makes the endospore coat an attractive route for heterologous antigen presentation.
We amplified the biofilm protein TasA, the endospore coat protein CotC and their promoters from the genome of B. subtilis NCIB3610. The metalloproteins, PbrR, GolB and SUP, are obtained by totally DNA synthesis. In order to verify the expression of proteins, we also utilized a constitutive promoter Pveg, which is a biobrick part in the biological parts of iGEM. Click here to see details about Pveg.
The following figures show our constructions.
Fig 8. The kit construction of our project.
We will then insert our genes of interest into the multiple cloning site of the shuttle plasmid pDG1730 which is shown in the picture. We insert our fusion genes between the amylase genes on the plasmid. By homologous recombination, our fusion genes will replace the amylase gene on the genome of B. subtilis.
Fig 9. The shuttle plasmid pDG1730
In order to test and verify whether homologous recombination is successful, we built the following three steps. The first step is the antibiotic selection. The second step is the bacterial PCR analysis. The last step is the amylase activity analysis. Because the inserted gene can replace the amylase gene on the genome of B. subtilis, the starch won’t be hydrolyzed and there won’t be the hydrolysis cycle on the plate when we apply iodine on the plate with starch.
The following picture shows the adhesion materials that we utilize to adhere the bacteria. They were obtained from School of Environment in Nanjing University. It has been proved by us that the four kinds of materials can tightly adhere microorganisms. The following picture shows the four kinds of materials that we originally obtained, of which we will choose the second generation plastic pellet. The second generation plastic pellet has the largest specific surface area so that it can adhere more microorganisms than the left three. It is the most sufficient and caters to our need to save energy.
Fig 10. Bacteria adhesion materials. 1. First generation plastic pellet 2. Second generation plastic pellet 3. Cyclic fiberfill 4. Chain-like fiberfill
By consulting an expert in School of Environment, Nanjing University, we designed a device which can hold the stuffing materials as well as the engineered microorganisms.
The parameters are as follows. The ratio of the device diameter to height is 1:7. The stuffing materials account for 1/3 of the device. The optimum current velocity is 1.5L/min. The following picture shows the blueprint of the device.
Fig 11. The blueprint of the device.
The device is made from Perspex, which is degradable and environmentally friendly. The stuffing materials with three kinds of engineered B. subtilis were put into three different devices so that we can retrieve the heavy metal respectively.
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