Team:HBUT-China/Parts
This section contains the promoter, RBS, BamHI restriction sites and terminators. We cut this part with the enzyme BamHI and inserted the nickel ion channel gene nikABCDE using the Gipson clone protocol.
This part contains the important gene ncrB, the important promoter pncrA, and the restriction site StuI. After expression of the protein NcrB, it will bind to pncrA, inhibit the function of pncrA, preventing the initiation of the expression of downstream genes. However, when nickel ions bind to the NcrB protein, the inhibition is released and the downstream gene begins to express. Here we designed the cleavage site StuI so that we can insert the reporter gene luxCDABE, again using the protocol of the Gipson clone.
This part carries the nickel ion channel protein gene nikABCDE (actually 5 genes, our figure is simplified for convenience). 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 which can provide energy for the transport of nickel ions. When ATP is not bound, the binding site of nickel ions 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 ions is transferred to the intracellular environment, thereby completing the transmembrane transport of nickel ions.
This section contains ncrB, pncrA and the reporter gene luxCDABE we inserted. luxCDABE is a bioluminescent gene. We measured the concentration of extracellular nickel ions by detecting the bioluminescence intensity of luxCDABE.
This part is our complete genetic line. By expressing nikABCDE, more nickel ions enter the cell, and more nickel ions will bind to NcrB, resulting in significantly increased expression of luxCDABE, and enhancing the bioluminescence value we could detect. As a result, the accuracy and sensitivity of our inspection system has been improved.
Parts
