Part:BBa_K3126023

hexa-His-nixA-TgMTP1t2-KanR

We connected hexa-his, nixA and TgMTP1t2 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 these three genes working in singles, pairs and the oinrigal yeast. (Part:BBa_K3126021 Part:BBa_K3126022Part:BBa_K3126020 Part:BBa_K3126019Part:BBa_K3126018).

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 pTEF1-hexa-His-tCYC1-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. Three proteins were expressed to enable engineered yeast to actively remove nickel ions in the environment.

In order to achieve this goal, we first use the surface display system of MFα1+hexa-his+ AGα1 to capture and bind nickel ions. We got the idea to reverse the application of protein purification (His-Tag), and instead use Hexa-his to attract and bind nickel ions to the cell surface. We used MFα1+hexa-his+ AGα1 to realize our idea. MFα1 can express a signal peptide that guides the fusion protein to the outside surface of the cell membrane, hexa-his and AGα1 together can express the Hexa-his—α-agglutinin fusion protein. It has a GPI anchor at the bottom that can attach the C-terminal of the α-agglutinin to the cell wall, and the Hexa-his will be placed on the N-terminal [1,2]. The reason we want to use the surface display system is that it has two advantages. Its function is only affected by the amount of protein expressed and it can bind nickel ions even after the yeast is dead.

Figure 1. Schematic diagram of surface display system of MFα1+hexa-his+ AGα1.

For the other path, 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 [3], and TgMTP1t2 is a channel protein that can transfer nickel ions from the cell’s internal environment to the vacuole [4]. 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 three sets of genes, our engineered yeast can actively bind or absorb nickel ions, and its tolerance to nickel ions is greatly increased.

Figure 2. Schematic diagram of function of the Composite Part (hexa-His-nixA-TgMTP1t2)

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.：

Figure 3. Absorption curve of simulated Ni²⁺ (15 mg/L) by engineering yeast with time.

We also compared its function with these three genes working in pairs (BBa_K3126021 BBa_K3126022):

Figure 4. Absorption curve of simulated Ni²⁺ (15 mg/L) by engineering yeast with time.

Figure 3. Absorption capacity of simulated Ni²⁺ (15 mg/L) by engineering yeast after 45 minutes treatment.

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=0.17  q_e=6.349 (mg/g)

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

K=0.298  q_e=3.701 (mg/g)

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 three genes were constructed together to express the strong absorption function of nickel ion, The equilibrium enrichment of genetically engineered yeast has been greatly improved, more than three times 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_k3126023 (hexa-His+nixA+TgMTP1t2) showed highest absorption efficiency, with a nickel ion removal efficiency of 80%, with the test concentration of nickel ions had being reduced from 15 mg/L to 2.6 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] Kuroda K , Shibasaki S , Ueda M , et al. Cell surface-engineered yeast displaying a histidine oligopeptide (hexa-His) has enhanced absorption of and tolerance to heavy metal ions[J]. Applied Microbiology & Biotechnology, 2001, 57(5-6):697-701.

[2] Kuroda K , Ueda M . Bioabsorption of cadmium ion by cell surface-engineered yeasts displaying metallothionein and hexa-His[J]. Applied Microbiology and Biotechnology, 2003, 63(2):182-186.

[3] 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.

[4] 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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 6538
  Illegal AgeI site found at 1049
  Illegal AgeI site found at 1310
  
• 1000
  COMPATIBLE WITH RFC[1000]