Part:BBa_K3126018
hexa-His-KanR
Hexa- His gene with TEF1 promoter and CYC1 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 ofSaccharomyces cerevisiae. This Composite part is composed by BBa_K3126014 (pTEF1-hexa-His-tCYC1) and BBa_K3126017 (pTEF2-KanR-tENO2). “hexa-His” is short for MFα1+hexa-his+ AGα1, which is the surface display system to capture and bind nickel ions. Kanamycin is a resistance gene, and was used to screen successfully homologous recombination of genes.
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.
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.：
The bioenrichment of nickel ions by yeast is a rapid reaction process. The Lagergren quasi-second-order dynamic model is used to describe:
t/qt=1/(k×qe2)+t/qe
qe---Enrichment of Ni2+ by yeast in absorption equilibrium (mg/g )
qt---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/qt=1/(k×qe2)+t/qe :
The absorption equilibrium qe and absorption equilibrium constant K were obtained by fitting the enrichment rate model:
K=0.1696 qe=3.663 (mg/g)
The absorption equilibrium qe and absorption equilibrium constant K were obtained by fitting the enrichment rate model:
K=0.3897 qe=2.44 (mg/g)
We can see that the equilibrium enrichment of genetically engineered yeast has been greatly improved.
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_K3126018 (hexa-His) showed higher absorption efficiency than original yeast, with the test concentration of nickel ions had being reduced from 15 mg/L to 8 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.
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 2842
Illegal AgeI site found at 1049
Illegal AgeI site found at 1310 - 1000COMPATIBLE WITH RFC[1000]
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