Team:ECUST/Fur Inverter
Description
Fur is a transcription factor that binds to fur box using Fe2+ as a helper repressor to inhibit gene transcription. The inhibition mechanism is shown in Figure 1. The Fe3+ is transferred into the cells by siderophore, and then Fe3+ is reduced to Fe2+. The Fe2+ binds to the Fur protein, and Fur-Fe2+ complex binds to the fur box to inhibit the expression of the export gene.
Design
As the siderophore is continuously secreted outside the cell, the concentration of the extracellular siderophore-Fe3+ complex is continuously increased. We want bacteria to be able to perceive the signal of high iron concentration (because this signal represents the degree of damage to the microenvironment that bacteria are living - rust), and the rust is converted from solid to ferric ions that are soluble and absorbed by the engineered E.coli.
That is to say, when the external Fe3+ concentration is high, the gene is in a condition of being suppressed. This is exactly the opposite of what we hope to be when the external Fe3+ concentration is high to trigger gene expression, so we need to introduce a reversal system. We reversed by introducing a lacI gene. As shown in Figure 2, when the concentration of external Fe3+ is high, the expression of the lacI gene is inhibited. At this time, the lacI gene cannot suppress the output, that is, the opening of the output gene is achieved.
Among them, the Fur protein binds to the 19-bp inverted repeat Fur box (GATAATGATAATCATTATC) with high affinity. There may be different positional relationships between the Fur box and the promoter, and this positional relationship results in a change in the iron concentration response threshold. In order to be able to adjust this threshold, we have designed three different positional relationships, as shown.
Plasmid construction
Result
Vector pET-28a(T7 promoter is replaced by J23119) is cut by BamH1 and Xho1, mCherry is amplified by PCR. Exogenous sequence（promoters with fur-box） and linearized vector are ligated by Ezmax.
The plasmid was transformed into E.coli DH5α, positive bacteria is cultured in LB medium adding with 0.1% kanamycin. Fluorescence intensity is measured at excitation wavelength of 587nm and emission wavelength of 610nm.
Fe-deficit medium is added 2,2’-bipyridine to final concentration of 200uM.
Fe-excess medium is added FeCl3 to final concentration of 100uM.
Bacteria is inoculated at 1% seed culture medium in Fe-deficit medium and Fe-excess medium and cultured for 12 hours. Then fluorescence intensity is measured.
Because fur2 has the lowest leakage expression of lacI under low Fe concentration and the highest expression under high Fe concentration, we choose fur2 promoter in our system.
Reference
1. PNAS November 28, 2017. 114 (48) 12785-12790 Sequential induction of Fur-regulated genes in response to iron limitation in Bacillus subtilis subtilis Hualiang
2. Chu et al. Microbial Cell Factories (2015) 14:37 A quorum sensing-based in vivo expression system and its application in multivalent bacterial vaccine Teng Chu, Chunshan Ni