Team:USP SaoCarlos-Brazil/Design
DESIGN
THE IDEA
Whether due to industrial waste poor management, mining activity or environmental disasters, several rivers and lakes throughout the country have heavy metal contamination, which can cause ecological imbalance, with social, economic and environmental impacts. Strong examples are the disruption of the Fundão dam Mariana in 2015 and the Brumadinho dam in Paraopeba river in January 2019. Up to 21 times the acceptable total lead and total mercury values were found in regions along the Paraopeba river. (IGAM, 2019) Then, we noticed the importance of finding a method for water decontamination of rivers and lakes with high levels of mercury. Currently, there are techniques applied to decontaminate water containing this heavy metal, such as mercury absorption by sulfuric acid functionalized activated carbon filter and ozone treatment. However, all available techniques are extremely expensive, making it impossible to apply them in large scale. Thus, our team found in synthetic biology and bioremediation the possibility of building an effective tool for solving this problem.
In view of the issue, our team began an intense literature search and thus we discover the Mer Operon. This operon is found in gram-negative and gram-positive bacteria, including Serratia marcescens, Pseudomonas putida, Staphylococcus aureus, Bacillus sp, Cupriavidus metallidurans, and Enterobacter, and gives resistance to mercury by encoding enzymes that are capable of changing the mercury oxidative state present in the environment. Hg+2 is absorbed by the cell and delivered to a NADPH-dependent enzyme that reduces the two Hg+2 electrons to Hg0, volatile and less toxic (Giovanella et al. 2010; Osborn 1997). Among the proteins expressed by this operon one caught our attention, Mer R, which binds to a mercury molecule and regulates the operon structural genes expression. (Nascimento 2013) Also, Mer Operon has a interesting promoter regulated by a mercury-binding protein (named MerR) that just allow the operon expression when its bonded to a Hg ion. Looking for other iGEM teams that had similar purposes to ours (heavy metal capture) we found iGEM10_Peking, which submitted the biobrick BBa_K346004 which consists in an engineered metal binding peptide from a Mer R protein homolog, having a duplicate metal binding domain. We chose this promoter to regulate the expression of our circuit and chose the chinese biobrick to compose our machinery due to Hg-binding enhancement when compared to non-modified MerR.
However, the metal-binding peptide bioaccumulates inside the bacteria and can cause its death precipitously, and in the Chinese biobrick the peptide's mercury uptake activity is restricted to the cytoplasm. Our team then came to the conclusion that the addition of a secretion mechanism to the mercury-capturing peptide could solve such deadlocks. Then, we began searching for secretion mechanisms applicable to our project. Studying the work of the Unicamp-EMSE_Brasil team, we found a secretion pathway, typical of gram-negative bacteria (type I secretion), consisting in four parts: the C-terminal alpha-hemolysin (HlyA) sequence, two specific translocating proteins HlyB and HlyD and the outer membrane protein TolC. The HlyB protein is responsible for the secretion process specificity as it recognizes the substrate by the secretion signal (such as HlyA). HlyD acts as an adapter protein, having a cytoplasmic domain followed by a membrane anchor and a large periplasmic domain. And finally, TolC, a protein that forms a channel through the outer membrane and periplasm toward the extracellular environment (Holland et al. 2005; Thomas et al. 2014). Thus, we decided to co-express the HlyB, HlyD and TolC proteins with the metal binding peptide as well as add the HlyA secretion signal to it.
So we come to an important question: How will we remove our bacteria from the water after treatment? This is a key point of our bioremediation project, because the use of genetically modified organisms in river water must be carefully planned to contain them, preventing the bacteria from spreading through natural ecosystems and causing environmental imbalance and human and animal health problems. Addressing this issue, we add to our circuit the expression a di-guanylate cyclase (DGC) that contains an active GGDEF domain responsible for signaling for biofilm formation. Biofilm is a biological community that is embedded in polymeric matrices produced by the bacteria themselves (Jenal and Malone, 2006; Hickman, Tifrea and Harwood, 2005). Thus, in our project, the formation of this structure on a substrate, the fiber of coconut, aims to facilitate the bacteria removal from water, since they will be trapped in the biofilm matrix, after they have fulfilled their goal of decreasing the concentration of soluble mercury. This material is cheap and abundant in Brazil and is often discarded despite its potential application. In addition, we thought it would be advantageous for our protein+Hg complex to get attached to the biofilm structure, facilitating its removal from the water being treated. Thereunto, knowing of the large amount of cellulose molecules that make up the E. coli biofilm, to ensure that once the peptide has been exported remains attached to the biofilm matrix, we add a cellulose binding anchor to it.
Therefore, we seek to develop a bioremediation method for mercury-containing contaminated waters using synthetic biology. In this project, is expressed in E. Coli a metal pickup chimera, containing secretion signal and cellulose anchor, and a GGDEF domain protein for biofilm induction, regulated by the same Mer promoter. In addition, they are co-expressed with proteins applicable to the secretion construct, HlyB, HlyD and TolC, regulated by a constitutive promoter, as we expect this machine to be ready for bacteria at all times.
THE CONSTRUCTION
The metal binding peptide used was obtained from the biobrick submitted by the iGEM10_Peking team, code Baa_K346004. Knowing that the C-terminal metal binding domain of the MerR homolog PbrR protein acts as a metal accumulator without the aid of the N-terminal DNA binding domain, this biobrick encodes a peptide that joins two metal binding domains copies to create a high performance metal binding peptide that consumes less energy. From the biobrick BBa_K1355001, deposited by the UFAM_Brazil team, we acquired the Operon’s Mer promoter sequence, regulated bu de mercury-binding protein MerR. The cellulose anchor (CBD) used was BBa_K1321340, submitted by the iGEM14_Imperial team, while the HlyA secretion signal sequence was obtained from biobrick BBa_K554002, deposited by the UNICAMP-EMSE_Brazil team. To be functional the secretion signal needed to be at the C-terminus of the protein to be secreted, so we constructed our metal scavenger chimera as follows: CBD + metal binding peptide + HlyA.
For secretion machinery, we used as parts HlyB (BBa_K554007), HlyD (BBa_K554008), TolC (BBa_K554009), also deposited by UNICAMP-EMSE_Brazil. These three proteins have been cloned into plasmid pSB1K3 and are regulated by the constitutive promoter BBa_J23100.
For biofilm induction, we chose from the literature three di-guanylate cyclases (DGCs): ydeH, originating from Escherichia coli DEC6A; wspR, from Pseudomonas fluorescens; and Q9X2A8, from Thermotoga maritima. In addition, searching in the Registry of Standard Biological Parts, we found a biobrick deposited by the iGEM12_HIT-Harbin team, code BBa_K748003, which codes for the protein YddV, a di-guanylate cyclase originating from the Escherichia coli O7: K1 genome, and added the same to our experiments to compare with the results of the other DGCs studied.
Thus, in E. Coli BL21 (DE3) was expressed the metal pickup chimera (CBD + metal binding peptide + HlyA), and a GGDEF domain-containing protein, both regulated by same Mer promoter. Besides, we also intend to express it regulated by the promoter Lac so its expression could be induced by IPTG.
As a consequence the bacteria would be able to produce our desired proteins without the metal presence and the metal capture machinery would be ready before it be exposed to Hg. For this, we would insert the construct into plasmid pACYDuet-1. However, we had no time to perform the necessary experiments with the Lac promoter.
THE EXPERIMENTS
The required sequences were synthesized by Twist Bioscience and cloning was performed by the method known as the Gibson Assembly, which allows multiple DNA fragments to be joined in a single isothermal reaction. This method uses three enzymatic activities: a 5 'exonuclease remove the 5' end nucleotides and exposes complementary sequences for pairing, a high fidelity DNA polymerase fills the gaps in the paired regions, and a DNA ligase covalently bind the DNA fragments. Gibson Assembly was chosen because it is a robust method for assembling DNA evenly and in the right order, resulting in a higher percentage of correct assemblies compared to the Golden Gate assembly, since the overlapping sequence of adjacent fragments is much longer in Gibson Assembly than is used in this second method.
After cloning the genes of interest, we need to check if our circuit really works, so we will do several experiments. To show that di-guanylate cyclases are effective in biofilm induction and to determine which protein is the best protein to compose our final circuit, we will clone the proteins separately in E. Coli BL21 (DE3), induce their expression and measure, by a technique that uses crystal violet, quantitatively biofilm formation. Also, we intend to co-transform our chassis with the pBS1K3 vector containing the secretion machinery and the pACYCDuet vector containing only our metal pickup chimera. The vectors were chosen considering their compatibility, pACYCDuet and pBS1K3 have replication origins from different families and have resistance to different antibiotics. However, we didn’t have enough time to the co-transformation experiment. With the complete circuit (chimera + di-guanylate cyclase), we made growth curves of the transformed bacteria in culture medium containing different concentrations of mercury and compare with the results obtained for the unmodified strain, evaluating if the expression of our chimera confers resistance to mercury and determining which are the most appropriate mercury concentrations to the other experiments.
With some conditions already determined by the previous experiments, we will start the experiments with the complete circuit (chimera + di-guanylate cyclase). In this phase of the experiments we seek to show that the initially idealized bioremediation is applicable. So, we will co-transform the bacteria with the vector containing the machinery needed for secretion and with the pACYCDuet vector containing the chimera and di-guanylate cyclase regulated by the same promoter. We will induce the expression of these proteins in medium containing a known mercury concentration and, a few hours later, separate the biofilm from the liquid medium by measuring the concentration of mercury that remained in solution and that was trapped in the biofilm. Also, we will test green coconut fiber as a substrate for the growth of this biofilm enriched with metal pickup chimeras. Thus, we will perform a last experiment in which we will insert green coconut fiber in a container containing the transformed bacteria and induce the chimeric protein expression and the protein responsible for biofilm formation. After biofilm is formed on the fiber, it will be transferred to a container containing culture medium with a known mercury concentration and after a few hours we will measure the concentration of this medium in contact with the fiber.
