Team:HIT-Harbin/Design

So as to test the working state of promoter when there is no dioxin, we designed this control circuit showed above. We replaced mdr83-805 by mdr521-805. Its sequence of dioxin receptor was eliminated. When it is stimulated by galactose in the environment, the circuit will express the DNA binding sequence which is independent of dioxin. It is especially for testing whether mdr521-805 can trigger transmembrane transportation. What’s more, Its localization effect as well as zero correction function can be tested.

In order to make yeast be capable of detecting dioxins in the environment, we designed the circuit above. TEF is the constitutive promoter which can activate downstream sequences to express dual domain protein, namely, lexA-DBD/mDR83-80. Comparing with other amplifiers, lexA can rapidly and efficiently induce the expression of downstream gene. At the meantime, we designed and applied the operator-lexop which possesses eight LEXAdbd binding sites, thus the catalytic effect of lexA operator is further improved. mDR83-805 can express mdr83-805（mouse dioxin receptor）. And after it combining intracellular dissociative dioxin, it will pass through karyotheca and combine with lexA operator to switch up downstream promoters and then activate the expression of green fluorescent protein downstream. Then we can know by observation whether there is dioxin in the environment or not.

Considering the amount of dioxin in the environment is in micro level or even trace level, when the concentration of dioxin decreases, those who can successfully combine with fusion protein lexadbd/mdr83-805 and then nucleic DNA will be less. Thus, we added a memory system to amplify the signal intensity by positive feedback regulation.

When there is a trace of dioxin, with the assistance of native intercellular hsp90 and arnt, the combination of fusion protein lexadbd/mdr83-805 and dioxin will activate the expression of florescent protein. Lex-A-DBD/mDR521-805 depicted in the picture is also dual domain protein but without binding site of dioxin hence cannot combine with dioxin. However, it can still pass through the nuclear membrane and combine with DNA to activate the expression of GFP. Such signal of the trace of dioxin can lead to the expression of a lot of GFP by amplification.

We combined yellow fluorescent protein induced by dioxins mentioned above together with DNA binding auxiliary sequence of lexAop and mdr521-805. Through the transformation of this genetic sequence, our device can express the yellow fluorescent protein massively and rapidly if the result of dioxin moleculesd detection is positive. In addition, as a result of the positive feedback effect, after the dioxin is removed, the device can still express the yellow fluorescent protein steadily, achieving the function of signal enhancement and memorization. In the absence of ligand, AhR is present in the cytosol in a complex with Hsp90, XAP2 and p23 proteins. Upon binding to a ligand, the AhR complex translocates into the nucleus and the AhR dissociates from Hsp90 complex to form a heterodimer with its partner molecule, Arnt. Thus, the formed AhR/Arnt heterodimer recognizes an enhancer DNA element designated xenobiotic responsive element (XRE) sequence located in the promoter region of CYP1A1gene, resulting in the enhanced expression of the gene[1].

Reference：[1] Functional role of AhR in the expression of toxic effects by TCDD

We combined yellow fluorescent protein induced by dioxins mentioned above together with DNA binding auxiliary sequence of lexAop and mdr521-805. Through the transformation of this genetic sequence, our device can express the yellow fluorescent protein massively and rapidly if the result of dioxin moleculesd detection is positive. In addition, as a result of the positive feedback effect, after the dioxin is removed, the device can still express the yellow fluorescent protein steadily, achieving the function of signal enhancement and memorization.

Here we add a rational design of cellular memory in yeast that employs autoregulatory transcriptional positive feedback .We combined yellow fluorescent protein induced by dioxins mentioned above together with DNA binding auxiliary sequence of lexAop and mdr521-805. Through the transformation of this genetic sequence, our device can express the yellow fluorescent protein massively and rapidly if the result of dioxin moleculesd detection is positive. In addition, as a result of the positive feedback effect, after the dioxin is removed, the device can still express the yellow fluorescent protein steadily, achieving the function of signal enhancement and memorization.

To make our green yeast multifunctional, we had searched thoroughly for projects dealingwith dioxin or polychlorinated biphenyl compounds. The good news was that we found out the project of Beijing Normal in 2008 was about the degradation of dioxins and PCBs. In their project biphenyl catabolic enzymes which can decompose PCBs was mentioned. This enzyme system is composed by four parts, namely, BphA, BphB, BpC, and BphD, as is shown in the picture. BphA is a kind of biphenyl dioxygenase, a riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4). It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1，catalyzes the initial 2,3-dioxygenation to obtain a 2,3-dihydrodiol compound. BphB is a kind of dihydriol dehydrogenase, that oxidize 2,3-dihydrodiol into 2,3-dihydrodiol. BPH3（2,3-Dihydroxybiphenyl dioxygenase）cleaves the dihydroxylated ring to produce (chlorinated) 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid with the presence of oxygen. It will be then hydrolyzed to (chlorinated) benzoic acid and 2-hydroxypenta-2,4-dienoate by a hydrolase, that is BPHD. At this moment, the toxicity of dioxin has been sharply decreased, which means we have accomplished our intension to degradation.

Meanwhile, we find binding protein Aga2 in the parts storage. This protein can specifically combine with aga1 protein on the cell wall of yeasts. The combination will lead to the immobilization of enzyme mentioned above.

For this section, we intended to bind seven peptide chains on the membrane so that dioxin in the domain can be degraded. However, we are also challenged by the problems like too large mass of protein to bind or the balance and regulation among different enzymes. Hence, this section of our project needed to be optimized in the future.

Dioxin is hard to dissolve in water but easy in organic solvents. However, yeasts live in the water. Thus, by improving the water-solubility of dioxin will largely increase the effect of our device. By looking through parts provided by previous teams, we found out AlnA(BBa_K398200), which is a kind of natural surfactant, an emulsifying protein present in the Alasan emulsifying complex which naturally occurs in Acinetobacter radioresistens. Nucleotide sequence optimized for expression in E.coli. Especially, we tagged secretion label on alnA protein in order to cut off secretion label of the fusion protein and secrete alnA outside of its cell with the effect of endoplasmic reticulum, so as to increase fat-soluble concentration around yeasts. Dissolution gradient will be formed for different concentration of dioxin from inside out, and then accomplish our goal of chemotaxis and enrichment of dioxin. Together with the presence of zif268-HIV binding domain, the expression of surfactant will be lengthened and well regulated.

In the project of HITGEM-2014, because of the new Gibson Method and a variety of problems in wet lab, we just finished the experiments and modeling proof for the first section. As for the following projects, only theoretical modeling and proof are realized. We will put a sound end to the following sections in the future.

About experiments, click here.

About modeling, click here.