Part:BBa_K3126021
hexa-His-nixA-KanR
We connected hexa-his 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_K3126018 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. The Composite part is used to homologous recombination in the chromosome of Saccharomyces cerevisiae. Kanamycin is a resistance gene, and was used to screen successfully homologous recombination of genes.
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.
We also use NixA to transfer nickel ions into cells. NixA is a channel protein that can transfer nickel ions from the external environment into the cell’s internal environment [3]. 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.
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
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_k3126021 (hexa-His-nixA-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] 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4690
Illegal AgeI site found at 1049
Illegal AgeI site found at 1310 - 1000COMPATIBLE WITH RFC[1000]
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