Cycle 1:Construct the Plasmid

1.1 Design

To enhance the bioleaching efficiency of Acidithiobacillus ferrooxidans, genetic engineering is essential. However, the molecular biology manipulation of A. ferrooxidans has proven to be intricate. The standard genetic engineering strategies, which include transformation, transduction, and conjugation, are not straightforward for this organism. The absence of suitable phages in the A. ferrooxidans genome rules out transduction as a method for introducing engineered plasmids. Moreover, previous attempts to prepare competent cells and to transform A. ferrooxidans have been unsuccessful, and the electroporation transformation method has yielded very low efficiency and inconsistent results^[1][2].

Given these challenges, conjugation stands out as the most promising approach. Despite its longer process and relatively lower efficiency, conjugation offers a simple and reliable means of plasmid transfer, making it a viable option for genetic manipulation of A. ferrooxidans. We have chosen E.coli SM10 as the donor strain for this process, leveraging its ability to transfer plasmids, such as pYYDT, which carries the mob gene, to recipient strains^[2].

In the genetic engineering of A. ferrooxidans, careful consideration must be given to plasmid selection markers. The acidic environment in which A. ferrooxidans thrives limits the use of resistance markers to a few options: mercury resistance (MerR), kanamycin resistance (KmR), streptomycin resistance (SmR), and chloramphenicol resistance (CmR). This constraint necessitates the selection of appropriate design methods for plasmid modification.

The pYYDT plasmid, a derivative of the pBBR series, Is an engineered vector that aligns with the RC10 assembly standard of synthetic biology. Its design, featuring a standard biological brick structure, facilitates genetic engineering. The presence of the mob gene from the broad-host-range plasmid pBBR1-MCS2 makes it suitable for conjugation. However, the overlap of kanamycin resistance (KmR) as a selection marker in both the donor strain E.coli SM10 and the pYYDT plasmid complicates the screening process for successful transformations. This overlap necessitates the modification of the pYYDT plasmid to differentiate between the donor and the recipient A. ferrooxidans strains that have been successfully transformed. This modification is critical for the successful application of pYYDT in the genetic engineering of A. ferrooxidans.

1.2 Build

For the construction of the plasmid pYDT, we employed a seamless cloning strategy. Initially, we tailored primers to match the sequences flanking the kanamycin resistance gene within the pYYDT plasmid. PCR amplification yielded a linearized version of the vector, from which the kanamycin gene was excluded. Subsequently, we designed primers for the streptomycin resistance gene, incorporating 20 bp homology arms for the linearized pYYDT at their extremities. These primers enabled the amplification of the streptomycin resistance gene, now equipped with the necessary pYYDT homology arms.

We then utilized homologous recombinase to seamlessly integrate the streptomycin resistance gene into the vector, creating the plasmid pYDT. This plasmid was introduced into E.coli DH5α competent cells, and correct integration was confirmed using colony PCR. The plasmid was purified and its accuracy was verified by agarose gel electrophoresis, with sequencing performed on plasmids exhibiting the appropriate band size.

Once the sequence was confirmed to be accurate, we prepared a glycerol stock by mixing equal parts of 700 μL glycerol with 700 μL bacterial solution, and stored the strains at -80°C for preservation.

1.3 Test

1.4 Learn

Having confirmed the sequence accuracy, we have successfully constructed the plasmid pYDT. This plasmid is now ready for conjugative transfer and subsequent modification of A. ferrooxidans, paving the way for further genetic engineering endeavors.

Cycle 2:Overexpress Diguanylate Cyclase (DGC)

2.1 Design

During the bioleaching process, A. ferrooxidans can form biofilms on the surfaces of minerals and accumulate cations such as Fe³⁺ and H⁺ , which are produced by the bacteria's own sulfur-iron metabolism, within the biofilms and Extracellular Polymeric Substances (EPS)^[1]. These cations are then used to leach the target minerals. Therefore, enhancing the ability of A. ferrooxidans to form biofilms can significantly improve its mining efficiency. Cyclic di-GMP (c-di-GMP), a widespread second messenger in bacteria, influences biofilm formation and dispersion. Numerous studies have demonstrated that higher intracellular concentrations of c-di-GMP correlate with increased biofilm production^[3]. By focusing on the metabolic pathway of c-di-GMP and overexpressing the c-di-GMP synthase DGC, we aim to increase biofilm production, enhance the interaction between bacteria and mineral surfaces, and subsequently improve leaching efficiency.

In order to further enhance the intracellular levels of c-di-GMP in the engineered bacteria, we have decided to clone the highly efficient DGC yedQ(BBa_K4242004) from E. coli, along with the DGCs AFE_0053(BBa_K5323000), AFE_1373(BBa_K5323999), and AFE_1379(BBa_K5323998) from the genome of A. ferrooxidans, into the expression vector pYDT. This approach aims to construct an efficient expression vector for DGCs and to conjugate this vector into A. ferrooxidans. Subsequently, we will measure the concentrations of biofilms and c-di-GMP, and screen for the most effective dynamic regulatory genes of c-di-GMP to guide our bioleaching operations.

Having previously obtained the plasmid expression vector containing biobricks, we opted for the double enzyme digestion method to construct the expression vector for this endeavor.

Firstly, we designed primers based on the sequences of four DGCs from the A. ferrooxidans genome. Through PCR amplification, we added restriction enzyme cutting sites at both ends of the genes. Using the double enzyme digestion method, we digested the target genes and the vector to create sticky ends, which were then ligated using a ligase to construct the expression vector. For yedQ, we initially optimized the codon usage for the species A. ferrooxidans, and the codon-optimized yedQ gene was synthesized by Tsingke Biotechnology Co., Ltd.. Subsequently, we directly obtained the yedQ gene with sticky ends from the synthesized product through double enzyme digestion and ligated it into the vector containing sticky ends to construct the expression vector.

2.2 Build

The basic procedurefor constructing the complete plasmid is as follows: Extract the plasmid vector pYDT and generate sticky ends through double enzyme digestion with restriction enzymes; obtain the target gene fragments with cutting sites either by PCR amplification or by synthesis from a biological company, create sticky ends on these target gene fragments through double enzyme digestion, and then ligate the target genes to the vector using a ligase to form the expression vector.

After obtaining the ligation products, they are transformed into E.coli DH5α competent cells. Colony PCR is used to verify and select bacteria that have been correctly transformed with the expression vector. The plasmid is then extracted and subjected to agarose gel electrophoresis. Plasmids with the correct band size are sequenced to confirm that the sequence is accurate. Once the sequence is confirmed to be free of errors,700 μL of glycerol is mixed with 700 μL of bacterial culture in a 1:1 ratio, and the mixture is stored at -80℃ for long-term preservation of the bacterial strain.

We plan to examine the results of overexpressing DGC using the expression vector in the model strain E.coli BL21, a prokaryotic organism, to determine the role of the expression vector in regulating biofilm formation. Initially, the expression vectors pYDT-0053, pYDT-1379、pYDT-1373、pYDT-yedQ and the empty vector pYDT will be transformed into E.coli BL21^[4]. Colony PCR validation will be used to select bacteria that have been correctly transformed with the expression vectors. In the correct transformants of E.coli BL21, we will assess the bacteria's ability to form biofilms and the levels of c-di-GMP within the cells (results can be found in the results section).

We will transform the correct expression vectors into the conjugative donor strain E.coli SM10 for conjugation experiments with the recipient bacteria A. ferrooxidans. Colony PCR will be used to verify and select A. ferrooxidans conjugants that have been correctly transformed with the plasmids, which will then be cultured on a larger scale. After extracting the genomic DNA from the conjugants, PCR will be performed to check the 16S rDNA sequence, and the sequences will be compared to confirm the acquisition of A. ferrooxidans conjugants. In the correct A. ferrooxidans conjugants, we will also test the ability to form biofilms and the levels of c-di-GMP within the cells (results can be found in the results section).

2.3 Test

During the construction of the expression vector, AFE_1360 exhibited cytotoxicity. Throughout the construction process, E.coli DH5α、BL21、TOP10 and SM10 transformed with pYDT-1360 were unable to grow normally. We suspect that overexpression of AFE_1360 in bacteria may produce a strong cytotoxic effect, severely inhibiting cell growth or even leading to cell death, thus making it unsuitable for overexpression. Consequently, we obtained four expression vectors: pYDT-0053、pYDT-1379、pYDT-1373 and pYDT-yedQ.. We successfully transferred these four expression vectors, along with the empty vector pYDT, into E.coli BL21 and A. ferrooxidans, and began to assess the impact of overexpressing DGC on bacterial biofilm formation and the levels of c-di-GMP within the cells.

2.4 Learning

After several failed attempts, we ultimately succeeded in obtaining the correct expression vectors. Following two consecutive failed conjugation experiments, we adjusted our experimental protocol. After making the necessary adjustments, we developed an efficient and reliable method to conjugate the plasmids into A. ferrooxidans, successfully achieving plasmid conjugation. These results are highly encouraging to us, marking the first experiment where an artificially designed biobrick-containing plasmid was conjugated into A. ferrooxidans. This experimental outcome opens up more possibilities for the future synthetic biology modification of A. ferrooxidans.

Cycle 3 Gold sensing Module

3.1 Design

In the realm of bioleaching, the natural environment's heavy metal stress often results in microorganisms exhibiting non-specific responses to a variety of metals. Typically, microorganisms do not selectively recognize or leach a specific metal, which greatly reduces the economic viability of bioleaching processes. Consequently, there is a significant opportunity in developing strategies for the specific recognition and recovery of precious metals such as gold.

We identified a gold-binding MerR family transcription factor, GolS, in Samonella sp., which enables the specific binding of Au[III] and triggers the transcription of downstream genes through the promoter P_gol. Leveraging this mechanism, we have designed a genetic circuit aimed at selectively recovering gold during bioleaching^[5].

We began by optimizing the golS gene's codon usage for A. ferrooxidans and synthesized the codon-optimized golS-P_gol gene fragment in collaboration with Tsingke Biotechnology Co., Ltd.. Employing a double restriction enzyme digestion method, we prepared both the target gene and the vector with compatible sticky ends. These were then ligated using DNA ligase to construct the expression vector pYDT-golS-P_gol. This engineered system is anticipated to enhance the precision and efficacy of gold recovery in bioleaching operations.

To assess the specificity and sensitivity of the gold-binding function, we introduced the gfp gene as a reporter. By measuring the fluorescence intensity of bacteria exposed to various Au[III] concentrations and different metals, we aimed to validate the system's selective response to Au[III].

For the construction of the pYDT-golS-P_gol-gfp expression vector, we again utilized the double restriction enzyme digestion method. The gfp gene, already present in our laboratory, was amplified via PCR with the introduction of restriction enzyme sites at both ends. Following this, the gfp gene and the pYDT-golS-P_gol vector underwent double enzyme digestion to create sticky ends. A DNA ligase was then used to ligate these fragments, successfully constructing the pYDT-golS-P_gol-gfp(BBa_K5323894) expression vector. This vector will facilitate the assessment of the system's specific response to Au[III] and inform the optimization of bioleaching conditions.

3.2 Build

The process of constructing the plasmid pYDT is methodical. Initially, we extract the plasmid vector pYDT and carry out a double restriction enzyme digestion to create sticky ends. Subsequently, we procure the target gene fragments with restriction sites; this can be done either by PCR amplification or by synthesizing them through a commercial service. Following this, the target gene fragments undergo double enzyme digestion to create sticky ends, which are then ligated to the vector with the aid of DNA ligase.

Once we have the ligation products, they are transformed into E.coli DH5α competent cells. Colony PCR is subsequently employed to verify and select bacteria that have been successfully transformed with the expression vector. The plasmid is extracted and its integrity is confirmed by agarose gel electrophoresis, checking for the correct band size. The plasmids that exhibit the appropriate band size are sequenced to ensure the sequence is correct. After successful sequence confirmation, we prepare a glycerol stock by mixing equal volumes of 700 μL glycerol with 700 μL bacterial culture, and the strains are stored at -80°C for long-term preservation.

Our subsequent plan involves assessing the specificity of Au[III] binding using the prokaryotic model strain E.coli BL21. For this, the expression vector pYDT-golS-P_gol-gfp is transformed into E.coli BL21, and colony PCR is used to select the successfully transformed colonies. In these confirmed transformants, we will evaluate the sensitivity and specificity of Au[III] binding, with results detailed in the subsequent results section.

3.3 Test

Through our experimental efforts, we have successfully acquired E.coli DH5α and BL21 strains that were correctly transformed with the expression vector pYDT-golS-P_gol-gfp. We proceeded to conduct tests aimed at evaluating the sensitivity and specificity of Au[III] binding. Given the time limitations and the extended duration associated with conjugative genetic engineering methods for A. ferrooxidans, we are actively engaged in experiments to conjugate the gold-binding expression vector into A. ferrooxidans. Our goal is to replicate the positive outcomes observed in E. coli strains within A. ferrooxidans as well.

3.4 Learning

Our experimental findings demonstrate that the E.coli BL21 strain, harboring the expression vector pYDT-golS-P_gol-gfp, is capable of specifically binding Au[III] and responding to even low concentrations of gold ions. This successful detection of the gene circuit's activity in the model strain BL21 indicates that it is performing as expected. Looking ahead, we intend to integrate the most efficient DGC (diguanylate cyclase) for biofilm regulation with this specific gold-binding gene circuit. The goal is to achieve an efficient and selective biorecovery system for gold.

Beyond the design of the engineered strains, we are also contemplating the practical applications of this project. A key consideration is the transition of this bioengineering from the lab to the field, particularly in recovering metals from abandoned tailings. To address this challenge, we have developed a safety suicide module. This module is a critical component in ensuring that our engineered organisms meet biosafety and ethical standards.

Cycle 4: kill switch

4.1 Design

To address the potential ecological risks of releasing genetically modified organisms into the environment, we are developing a kill switch. This safety feature is designed to ensure that the engineered bacteria only survive under specific conditions, particularly in the presence of Au[III] ions. To implement this, we have integrated a classical inverter gene circuit, which includes the cI repressor from the E.coli λ phage and its associated promoter P_R, into the gold-binding gene circuit. Additionally, we have introduced the toxic protein MazF, creating a system that ensures the engineered bacteria can only thrive in environments rich in Au[III].

The construction of the suicide gene circuit involved a double enzyme digestion method. We amplified the cI-PR segment, adding restriction sites via PCR. The cI-P_R gene segment and the vector pYDT-golS-P_gol were then digested with restriction enzymes to produce compatible sticky ends, which were subsequently ligated to create the expression vector pYDT-golS-P_gol-cI-P_R(BBa_K5323892).

For the integration of the mazF gene, which encodes the toxic protein, we initially optimized its codon usage to match that of A. ferrooxidans. The codon-optimized mazF gene fragment was synthesized by Tsingke Biotech Co., Ltd. Following this, we employed the double enzyme digestion method to create sticky ends on the mazF gene and the vector, which were then ligated to form the final expression vector pYDT-golS-P_gol-cI-P_R-mazF(BBa_K5323893). This comprehensive design ensures that our engineered bacteria are not only equipped for the task of gold recovery but also safeguarded with a built-in mechanism to contain their survival to controlled conditions.

4.2 Build

The fundamental procedure for constructing a plasmid is as follows: We initiate the process by extracting the plasmid vector pYDT and proceed with double enzyme digestion to create sticky ends. The target gene fragment, equipped with restriction enzyme sites, can be acquired through PCR amplification or by synthesis provided by a biotechnology company. Following this, the target gene fragment undergoes double enzyme digestion to generate compatible sticky ends, which are then ligated to the plasmid vector using a ligase.

After obtaining the ligation product, it is transformed into E.coli DH5α competent cells. We then conduct colony PCR verification to select for colonies that have correctly incorporated the expression vector. The plasmid is extracted and subjected to agarose gel electrophoresis for analysis. Those plasmids exhibiting the correct band size are sequenced to ensure accuracy. With the sequence confirmed, a mixture of 700 μL glycerol and 700 μL bacterial solution is prepared at a 1:1 ratio and stored at -80℃.

Our subsequent plan involves testing the suicide switch functionality using the prokaryotic model strain E.coli BL21. The expression vector pYDT-golS-P_gol-cI-PR-mazF will be introduced into E.coli BL21, and colonies containing the correct expression vector will be identified through colony PCR verification. In these successfully transformed E.coli BL21 strains, we will perform induced expression experiments to assess the suicide plasmid's effectiveness. (results will be found in the results section).

4.3 Test

After performing the experiments, we successfully obtained E.coli DH5α and BL21 strains that were correctly transformed with the expression vector pYDT-golS-P_gol-cI-P_R-mazF. We then proceeded to test the sensitivity and specificity to Au[III] ions. However, due to time constraints, we were unable to obtain A. ferrooxidans zygotes containing the expression vector. We anticipate observing positive experimental results in subsequent supplementary experiments.

4.4 Learning

At this stage, we have delineated the complete construction process of our project. By seamlessly integrating the aforementioned modules, our team is committed to offering a sustainable and efficient methodology for the leaching of precious metal gold utilizing A. ferrooxidans. This innovative approach is designed to not only bolster the recovery rate of valuable resources but also to place a premium on environmental safety. Consequently, it stands as a promising solution for bioremediation and bioleaching applications, aligning with the growing demand for eco-friendly technologies.

Our research provides a foundational framework that paves the way for the further advancement of microbial mining technology. The successful transformation of A. ferrooxidans with artificially designed biobrick plasmids marks a significant step forward. It streamlines the synthetic biological modification of this strain and supports the resource extraction industry in embracing more sustainable practices. This development is poised to have a substantial impact, potentially revolutionizing the industry by offering an environmentally conscious alternative to traditional mining methods.

Reference

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[2] Chen J, Liu Y, Diep P, et al. Genetic engineering of extremely acidophilic Acidithiobacillus species for biomining: Progress and perspectives[J]. Journal of Hazardous Materials, 2022, 438: 129456.
[3] Jenal U, Reinders A, Lori C. Cyclic di-GMP: second messenger extraordinaire[J]. Nat Rev Microbiol, 2017,15(5):271-284.
[4] Sand, W. and Gehrke, T. (2006) Extracellular polymeric substances mediate bioleaching ⁄ biocorrosion via interfacial processes involving iron(III) ions and acidophilic bacteria. Res Microbiol 157, 49–56.
[5] Wei W, Zhu T, Wang Y, et al. Engineering a gold-specific regulon for cell-based visual detection and recovery of gold[J]. Chemical Science, 2012, 3(6): 1780-1784.