Team:HIT-Harbin/Design

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.

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.

Reference:

1.Mimura J, Fujii-Kuriyama Y. Functional role of AhR in the expression of toxic effects by TCDD[J]. Biochimica et Biophysica Acta (BBA)-General Subjects, 2003, 1619(3): 263-268.

2.Denison M S, Heath-Pagliuso S. The Ah receptor: a regulator of the biochemical and toxicological actions of structurally diverse chemicals[J]. Bulletin of environmental contamination and toxicology, 1998, 61(5): 557-568.

3.Toren A, Segal G, Ron E Z, et al. Structure–function studies of the recombinant protein bioemulsifier AlnA[J]. Environmental microbiology, 2002, 4(5): 257-261.

4.Furukawa K, Fujihara H. Microbial degradation of polychlorinated biphenyls: biochemical and molecular features[J]. Journal of bioscience and bioengineering, 2008, 105(5): 433-449.

5.Whitelaw M L, Göttlicher M, Gustafsson J A, et al. Definition of a novel ligand binding domain of a nuclear bHLH receptor: co-localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor[J]. The EMBO journal, 1993, 12(11): 4169.