Part:BBa_K3126020

TgMTP1t2-KanR

TgMTP1t2 gene with TDH3 promoter and PGK1 terminator, using KanR as resistance cassette. We characterized the ability to adsorb nickel ions of Saccharomyces cerevisiae which expressed this part.

Usage and Biology

The sludge in the activated sludge process consists of a variety of microorganisms, which originally include yeast. Due to its excellent heavy metal tolerance, our project aim to develop engineering yeast to absorb nickel ions. The Composite part is used to homologous recombination in the chromosome of Saccharomyces cerevisiae.

We use TgMTP1t2 to transfer nickel ions from the cells into the vacuoles. TgMTP1t2 is a channel protein that can transfer nickel ions from the cell’s internal environment to the vacuole [1]. Kanamycin is a resistance gene, and was used to screen successfully homologous recombination of genes. Because the nickel ions will do harm to the yeast if they remain in the internal environment, we want to move them into the vacuole which can safely store more nickel ions.

Under the coordination of these sets of genes, our engineered yeast can actively bind or absorb nickel ions, and its tolerance to nickel ions is greatly increased.

Figure 1. Schematic diagram of function of the Composite Part (TgMTP1t2-KanR)

Result

Here is the curve comparing with original Saccharomyces cerevisiae. If you want to know more about our experimental methods, please click here https://2019.igem.org/Team:HBUT-China/Notebook .

Figure 2. Engineering yeast comparing with original yeast

Conclusion

The absorption abilities of engineered yeast and original yeast were compared, and the results indicated that among all of the engineering yeast, the S.cerevisiae/BBa_k3126020 (TgMTP1t2-KanR) showed higher absorption efficiency than original yeast. Our results proved that this composite part is a biologically functional composite part.

Potential applications

In the future, this Composite part can be used to be introduced to other species of microorganisms to improve their nickel ion absorption capacity.

References

[1] Persans, M. W., Nieman, K., & Salt, D. E. (2001). Functional activity and role of cation-efflux family members in Ni hyperaccumulation in Thlaspi goesingense. Proceedings of the National Academy of Sciences, 98(17), 9995-10000.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 726
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2200
  
• 1000
  COMPATIBLE WITH RFC[1000]