Bioleaching Plasmid (6186 bp)

Lithium Detection Plasmid (6186 bp)

Arsenic Detection Plasmid

The parts comprising the bioleaching plasmid included the plasmid backbone itself (BBa_J428326) and two main other enzyme parts.

The Lithium detection plasmid included the backbone (BBa_J428326) combined with the lithium detector and a Kill switch.

The Arsenic detection plasmid included the backbone (BBa_J428326) combined with the lithium detector and a Kill switch.

• Tetrathionate Hydrolase:

  The Tetrathionate Hydrolase, an enzyme which catalyses tetrathionate hydrolysis, plays a distinctive role in the acidophilic iron and sulphur oxidizing bacterium A. ferrooxidans. It is responsible for the dissimilatory of sulphur oxidation to generate sulphate, elemental sulphur, thiosulfate. This part successfully combined tetrathionate Hydrolase protein with a constitutive promoter, a ribosome binding site (BBa_J61100) and a terminator (BBa_B0010).

  

• High potential iron Sulphur protein (HiPiP) and pH Resistance gene:

  The HiPiP, as its name suggests, has a high redox potential hence is effective in catalysing the oxidation reaction which takes place. With the lithium biomining process involving oxidation reactions with sulphide minerals, this part plays an crucial role in enhancing the E.coli bacteria lithium recycling activity. During the lithium bioleaching process, it is normal for low pH levels to arise due to the increased concentration of hydrogen ions thus the need for the pH resistance gene. This ensures the growth and metabolism of our strain of bacteria in the bioleaching environment. Synthesizing this complete part involved the use of two promoters, two ribosome binding sites (BBa_J61100) and three terminators (BBa_B0010), as well as the HiPiP and the pH resistance gene.

  

• The HiPiP, as its name suggests, has a high redox potential hence is effective in catalysing the oxidation reaction which takes place. With the lithium biomining process involving oxidation reactions with sulphide minerals, this part plays an crucial role in enhancing the E.coli bacteria lithium recycling activity. During the lithium bioleaching process, it is normal for low pH levels to arise due to the increased concentration of hydrogen ions thus the need for the pH resistance gene. This ensures the growth and metabolism of our strain of bacteria in the bioleaching environment. Synthesizing this complete part involved the use of two promoters, two ribosome binding sites (BBa_J61100) and three terminators (BBa_B0010), as well as the HiPiP and the pH resistance gene.

  

• Arsenic Detector:

  The arsenic detector part consists of the constitutive ParsWT promoter and the reporter gene, amilGFP (BBa_K592010/BBa_K1033931 ). When arsenite is present in the environment, they bind to the ArsR protein and this action further enables the expression of the amilGFP reporter gene. The bacteria display a vibrant yellow colour upon expression of the chromoprotein, and to ensure that transcription ceases after gene expression, we incorporated the terminator (BBa_B0010) at the end of the genetic sequence.

  

  Kill Switch:

  Despite the containment of the bacteria in hydrogel, it is essential that they pose no threat to the external environment should they escape. This is why a kill switch is necessary. It comprises of an inducible promoter (BBa_I765001), which is activated in the presence of UV light. T4 endolysin (BBa_K112806), a lysozyme from bacteriophage T4, is then transcribed which catalyses the hydrolysis of peptidoglycan in the bacterial cell wall which results in the rupturing and destruction of the cell. A ribosome binding site (BBa_J61100) and a terminator (BBa_B0010) was also included in the design of this composite part which acts as an effective kill switch.

  

  Reference:

  [1] V., B. and S., S.S. (2022) Indicator Minerals, Pathfinder elements, and portable analytical instruments in Mineral Exploration Studies [Preprint]. doi:10.20944/preprints202202.0272.v1.

• Lithium Detector

  The lithium detector part consists of an nhaA-I promoter and the reporter gene, eforRed (BBa_J435099). The nhaA-1 gene encodes a lithium antiporter protein responsible for the active transport of lithium ions into the cell, followed by their excretion to maintain cellular homeostasis. Upon exposure to lithium ions, the nhaA-I promoter is transcribed, initiating the expression of the chromoprotein reporter gene, eforRed. Consequently, the bacteria express a red color, providing a visual indicator of the presence of lithium. To ensure effective translation of the reporter gene, a ribosome binding site (BBa_J61100), RBS, is positioned between the promoter and the reporter. This RBS facilitates efficient translation of eforRed, enabling the synthesis of the reporter protein. Additionally, a terminator sequence (BBa_B0010). is incorporated to appropriately end the transcriptional process.

  

  Kill Switch:

  Despite the containment of the bacteria in hydrogel, it is essential that they pose no threat to the external environment should they escape. This is why a kill switch is necessary. It comprises of an inducible promoter (BBa_I765001), which is activated in the presence of UV light. T4 endolysin (BBa_K112806), a lysozyme from bacteriophage T4, is then transcribed which catalyses the hydrolysis of peptidoglycan in the bacterial cell wall which results in the rupturing and destruction of the cell. A ribosome binding site (BBa_J61100) and a terminator (BBa_B0010) was also included in the design of this composite part which acts as an effective kill switch.