Difference between revisions of "Part:BBa K2652006"

Revision as of 09:42, 20 October 2019

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