This year, our team NTHU_Taiwan would like to introduce innovative ways to increase the yield of quantum dots by our gene design. They are members of BioSQUAD, JM109, JM109 with yhaO, JM109 with MntH, JM109 with MT2 and JM109 with CadA. With this powerful squad, we obtain a much higher quantum dots amount.
Promoter CadA (BBa_K1724000) allows us to enhance the proteins production in the environment contains cadmium ions. Gene MntH(BBa_K1526007) allows the concentration of cadmium ions be almost equal with extracellular and intercellular one. MT2 lower the toxicity of cadmium ions, which is a heavy mental by using a peptide to combine with ions thus form a chelate. Last but not least, yhaO(BBa_K2242233) can turn cysteine into hydrogen sulfide, which is a better ingredient for quantum dots synthesis.
With those four gene below, we construct the gene function and quantum dots production pathway
1. Successfully cloning all of gene in JM109.
2. Successfully cloning of two plasmid experiment (cada-mnth/cada-MT2) and (cada-mnth / cada-yhaO).
3. Successfully verify each gene is functional.
4. Verify MT2's efficiency against cadmium ions.
5. Verify yhaO can help produce QD.
Part one: Gene CadA
Among the large group of metals, there are few essential metals like zinc and manganese which are micronutrients used in redox processes, regulation of the osmotic pressure, and also enzyme components. Besides them most of all other non-essential metals are potent to have toxic effect. There is presence of abundance amount of both toxic and non-toxic metal ions in the nature, but industrial use of heavy metals leads to serious environmental issues.
The use of metal-resistance bacteria can help to remove metal ions from defiled water. Understanding the regulation of heavy metal resistance could be useful for biological waste treatment and to flourish the ecosystem, we planned to use bacteria for quantum dots production. Bacterial metal resistance systems are regulated by transcriptional factors from the MerR family. CadA sequence has a histidine-rich N-terminal extension which helps in the binding of metal ion. Based on the recent studies CadA may play a broader role in cadmium resistance in gram-negative bacteria, so following this trend we did the general DNA manipulations and DNA sequencing. Standard recombinant DNA techniques were carried out for restriction endonuclease digestion, ligation, transformation of plasmid DNA, and isolation of total DNA.
Part two: Gene MntH
Our goal is to catch cadmium metal ions from waste water using genetically engineered E.coli. Due to the toxicity of natural heavy metals, E.coli resist internalization of heavy metal ions under normal conditions. However, sometimes heavy metals might be accidentally pumped inside the cell (e.coli) by certain channel protein. Cadmium enters bacterial cells by the transport systems for essential divalent cations such as Mn with charge of 2+ or Zn with charge of 2+, which carries same valance electron as cadmium. This false recognition leads to pumping of heavy metal (Cd) into the cell.
MntH protein is a member of the natural resistance-associated macrophage proteins (NRAMP) family of metal ion transporters. The protein has 11 putative membrane spanning alpha helices. This permease is involved in the uptake of metal with charge of 2+.The protein is mainly pump Mn2+ and Fe2+ but also have a affinity to cadmium.
Part three: Gene MT2
Although we can allow lots of cadmium enter into JM109 after cloning of MntH gene, it will cause our E.coli to die of high concentration of cadmium toxin. We must find something to protect E.coli from cadmium and maintain cadmium inside the E.coli at the same time.
Lots of peptide can combine with heavy metal such as GSH (glutathione) , we choose metallothionein because it is widely used protein to help bacteria against cadmium, and some papers also indicate that MT2 can cooperate well with MntH protein.
We use colocasia esculenta metallothionein type 2 as our MT2 out of plenty of metallothionein, colocasia esculenta is a plant that owns the ability to sustain heavy metal environment. With this property, we try to apply it in our gene design.
Metallothionein (MT) is low-molecular weight, cysteine-rich protein in Cd toxicology. Cd-bound to MT is responsible for Cd accumulation in tissues and the long biological half-life of Cd in the body. Induction of MT protects against acute Cd-induced lethality, as well as acute toxicity to the liver and lung.
MT gene is proof to be functional in bacteria too. Normally one MT can combine with seven cadmium by its thiol group, which can not only decrease the cd toxin to JM109 but make cd stay inside the e.coli.
Part four: Gene yhaO
In order to introduce a synthetic biology and energy-saving way to manufacture quantum dots, we must increase the relative fluorescence value and the quantum rate. We use a gene named "yhao" as L-Cysteine desulfhydrases for enhancing the producibility.
In our experiment, we use cysteine and cadmium chloride as our precursor. Cysteine is a relatively common amino acid in biology system. However, it is complex molecule compared to hydrogen sulfide which is the better choice for sulfur resource. The reason that we add cysteine instead of hydrogen sulfide also related to the toxicity of hydrogen sulfide. Still less, hydrogen sulfide exist in gas phase with our experimental environment.
The yhao gene we used has been specified in E. Coli MG1655that it can use a L-Cysteine desulfhydrases in a pathway that include a redox system that can transform the cysteine into hydrogen sulfur.
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