Part:BBa_K3126022

nixA-TgMTP1t2-KanR

We connected TgMTP1t2 and nixA with their promoters and terminators, and used KanR as resistance cassette. We characterized the ability to adsorb nickel ions of Saccharomyces cerevisiae which expressed this part. We also compared its function with those two functional genes working separately (Part:BBa_K3126020 Part:BBa_K3126019).

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. This Composite part is composed by pGPM1-nixA-tTDH1-pTDH3-TgMTP1t2-tPGK1-pTEF2-KanR-tENO2. Kanamycin is a resistance gene, and was used to screen successfully homologous recombination of genes. The Composite part is used to homologous recombination in the chromosome of Saccharomyces cerevisiae. Two proteins were expressed to enable engineered yeast to actively remove nickel ions in the environment.

We found two proteins, NixA and TgMTP1t2. We also use NixA to transfer nickel ions into cells, and then use TgMTP1t2 to transfer nickel ions from the cells into the vacuoles. NixA is a channel protein that can transfer nickel ions from the external environment into the cell’s internal environment [1], and TgMTP1t2 is a channel protein that can transfer nickel ions from the cell’s internal environment to the vacuole [2].

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 (nixA-TgMTP1t2－KanR)

If you want to know more about our experimental methods, please click here https://2019.igem.org/Team:HBUT-China/Notebook

Result

We carried out absorption experiments with the original yeast and the genetically engineered yeast we constructed at the same time and made sure that the other conditions were exactly the same. The experimental results are as follows.：

We also compared its function with those two functional genes working separately (BBa_K3126020 BBa_K3126019).

The bioenrichment of nickel ions by yeast is a rapid reaction process. The Lagergren quasi-second-order dynamic model is used to describe:

t/q_t=1/(k×q_e²)+t/q_e

qe---Enrichment of Ni²⁺ by yeast in absorption equilibrium (mg/g )

q_t---Enrichment of Ni²⁺ by yeast at t time (mg/g )

K--- Absorption constant (mg/L)

Absorption curve of Ni²⁺ (15 mg/L) by genetically engineered yeast with time:

Linear fitting by formula t/q_t=1/(k×q_e²)+t/q_e :

The absorption equilibrium q_e and absorption equilibrium constant K were obtained by fitting the enrichment rate model:

K=0.135  q_e=5.136 (mg/g)

The absorption equilibrium q_e and absorption equilibrium constant K were obtained by fitting the enrichment rate model:

K=0.2817  q_e=3.3245 (mg/g)

The absorption equilibrium q_e and absorption equilibrium constant K were obtained by fitting the enrichment rate model:

K=0.4694  q_e=3.201 (mg/g)

We can see that the proteins that connect the two genes together can adsorb nickel ion more effectively than a single gene expression proteins can adsorb nickel ion. The equilibrium enrichment of genetically engineered yeast has been greatly improved, more than twice as much as the 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_k3126022 (nixA-TgMTP1t2) showed higher absorption efficiency than original yeast, with the test concentration of nickel ions had being reduced from 15 mg/L to 5 mg/L after absorption for 45 min. 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] Deng, X., He, J., & He, N. (2013). Comparative study on Ni2+-affinity transport of nickel/cobalt permeases (NiCoTs) and the potential of recombinant Escherichia coli for Ni2+ bioaccumulation. Bioresource technology, 130, 69-74.

[2] 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 2574
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4048
  
• 1000
  COMPATIBLE WITH RFC[1000]