Part:BBa_K2652006
Improved by Fudan iGEM 2024
In order to import nickel ion into the E.coli, we choose to adopt nikABCDE into our design.
Improved part
Our improved part is BBa_K5115082 (ribozyme connected nik). The improved part is combines BBa_K5115077(ribozyme+RBS+nikA+stem-loop), BBa_K5115078(ribozyme+RBS+nikB+stem-loop),BBa_K5115079(ribozyme+RBS+nikC+stem-loop), BBa_K5115080(ribozyme+RBS+nikD+stem-loop)and BBa_K5115081(ribozyme+RBS+nikE+stem-loop). We introduced this ribozyme-assisted polycistronic co-expression system from 2022. By inserting ribozyme sequences between CDSs in a polycistron, the RNA sequences of Twister ribozyme conduct self-cleaving, and the polycistronic mRNA transcript is thus co-transcriptionally converted into individual mono-cistrons in vivo.
With this design, we achieve co-expression of BBa_K5115077(ribozyme+RBS+nikA+stem-loop), BBa_K5115078(ribozyme+RBS+nikB+stem-loop),BBa_K5115079(ribozyme+RBS+nikC+stem-loop), BBa_K5115080(ribozyme+RBS+nikD+stem-loop)and BBa_K5115081(ribozyme+RBS+nikE+stem-loop) at similar level.
The ribozyme-connected nikABCDE operon we use facilitates the import of nickel ions, essential for sustaining Ni/Fe hydrogenases in E. coli under anaerobic conditions, where nickel is required for hydrogenase activity. The NikABCDE transporter consists of five proteins that work together to import nickel from the environment, despite its low natural availability [1]. In our design, the ribozyme linkage aids in fine-tuning the expression of the nik operon, ensuring efficient nickel uptake for cellular processes.
Nickel ion channel protein gene
NikA is in the periplasm between the cell membrane and the cell wall. It can capture and bind to free nickel ions and transport it to NikB and NikC located on the cell membrane. NikB and NikC have binding sites for nickel ions. NikD and NikE can be combined with ATP provides energy for the transport of nickel ions. When ATP is not bound, the binding site of nickel ion is exposed to the outside of the cell membrane. After ATP binding, the protein structure of NikB and NikC changes, and the binding site of nickel ion is transferred to the intracellular measurement, thereby completing the transmembrane transport of nickel ions.
Improvement
- Group: iGEM Team HBUT China 2019
- Author: Weiwei Li
Background
In last year's Nickel Hunter 2.0, we found that the nikABCDE system present in other strains of E. coli belongs to the ATP-binding cassette (ABC) protein family, and contains five proteins that can transport the hydrolyzed ATP to the transmembrane transport of nickel ions. NikB and NikC are two transmembrane proteins that form the transmembrane core of the transport system. NikA is a periplasmic binding protein (PBP) that transmits captured nickel ions to the NikBC core. NikD and NikE act as two cytoplasmic proteins. The signal-to-noise ratio of the system is increased by the nickel ion channel protein, and the detection accuracy is improved.
Inspiration
His-tag histidine tags can interact with various metal ions, including Ca2+, Mg2+, Ni2+, Co2+, etc. Among them, nickel ions are most widely used. It fuses multiple histidine strands (commonly 6 histidine) at the end of the recombinant protein. By using this histidine peptide segment to chelate with divalent metal ions (nickel, zinc, etc.), nickel ions can be adsorbed.
Methods
This year we added His-tag sequences to both ends of nikABCDE, which is like adding a pair of hands to last year's nikABCDE proteins to optimize it.
Protocols
1.Digested the pSB1C3-BBa_K2652001 circular plasmid with BamHI.
2.Amplified our target gene BBa_K3126026（His-Tag-nikABCDE- His-Tag） using PCR technology.
3.Recovered the digested part and inserted BBa_K3126026（His-Tag-nikABCDE- His-Tag）into pSB1C3-BBa_K2652001 by seamless cloning, and the mixed system was reacted at 37 ° C for 30 minutes.
4.Converted all of the products of the above seamless cloning system to DH5α.
5.Applied DH5α to the plasmid to a plate containing chloramphenicol resistance.
6. Culture E.coli for a period of time, measure its OD600
7. Convert the required dry weight volume according to OD600
8. Add the corresponding volume of bacterial solution into 50mL centrifuge tube, centrifugate at 8000 rpm for 12 min, discard the supernatant and enrich the cells (more than 30mL is divided into two centrifuges);
9. Prepare 6 bottles of 50mL 30mg / L nickel ion solution, put the E.coli into the solution respectively, and culture them in a shaker at 37 ℃.
10. Take 1.5mL solution every 2min in the centrifuge tube, take 5 times, and then take 3 times every 5min;
11. Using dimethylglyoxime spectrophotometry to measure the nickel ion concentration of the above samples;
12. Draw the relation curve about time and nickel ion concentration.
Results
Two kinds of engineering yeas were cultured in 30 mg/L nickel ion solution. Within 100 minutes, it can be seen from the figure that the improved engineered yeast has a stronger nickel adsorption ability than the original yeast.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1560
Illegal AgeI site found at 3561 - 1000COMPATIBLE WITH RFC[1000]
- ↑ Dosanjh, N. S., & Michel, S. L. (2006). Microbial nickel metalloregulation: NikRs for nickel ions. Current opinion in chemical biology, 10(2), 123–130. https://doi.org/10.1016/j.cbpa.2006.02.011
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