Team:Bilkent-UNAMBG/Description

Smelling Cancer

Cancer is one of the most common and lethal disease in the world and it is always highlighted that early diagnosis is crucial in terms of cancer treatment. It is often invasive and painful to diagnose cancer with the existing methods. At this point, breath test appears as a promising non-invasive and real-time technique which allows the monitoring of metabolic status.

Volatile organic compounds(VOCs) in the exhaled breath provide in vitro detection, classification and discrimination of diseases and microorganisms. Concentration of specific VOCs in the exhaled breath can either increase or decrease depending on the type of diseases and microorganisms.

In this project, our goal is to produce a prototype which can detect and discriminate four most common cancer types: lung, breast, colorectal and prostate. In order to achieve this, we will engineer an artificial plasmid including promoter and reporter genes that are specific to a particular VOC. It is expected that when that particular VOC is given into the environment, bacteria will express reporter gene hence we will get some signal. After achieving getting the signal for a particular VOC, we aim to construct logical gates and circuits by using CRISPRi system on plasmids to create well accoutred bacteria which will be able to differentiate these four cancer types.

Our future goal is to transform our prototype into a kit. So that, diagnosis of cancer will be simpler and non-invasive.

tetR is produced under control of constitutively active promoter, which is indicated in gray. Since TetR is produced continuously, plteto1 is inhibited, unless Atc is present in the environment. When Atc is present, promoter plteto is cleared and XylrR can be expressed, since XylR is on the downstream sequence. Output is seen as sfGFP only if o-xylene is present in the environment. When present, o-xylene and XylR activaes Pu promoter and sfGFP is produced.

tetR is produced under control of constitutively active promoter, which is indicated in gray. Since TetR is produced continuously, plteto1 is inhibited, unless Atc is present in the environment. When Atc is present, promoter plteto is cleared and ChnR can be expressed, since ChnR is on the downstream sequence. Output is seen as sfGFP only if cyclohexanone is present in the environment. When present, cyclohexanone and ChnR activaes pchnb promoter and sfGFP is produced.

Since lacI is produced under the constitutively active promoter, indicated in gray, promoter pllac/ara1 is always inhibited, unless IPTG is added into the environment. When added, it removes lacI and the promoter now can enable the production of ChnR and XylR. If there is cyclohexanone in the environment, ChnR and cyclohexanone activates pchnb promoter to produce sgRNA. The produced XylR can activate the Pu promoter if and only if o-xylene is present in the environment. When present, they activate Pu promoter and dCas9 production. The produced sgRNA and dCas9 now come together and find the sequence of pllaco, thus enable sfGFP production, which is the output.

References

http://www.uniprot.org/uniprot/Q9R2F5

Abril, Maria-Angeles, Martin Buck, and D Juan L. Ramos. "Activation of the Pseudomonas TOL Plasmid Upper Pathway Operon." THE JOURNAL OF BIOLOGICAL CHEMISTRY (1991): 15832-583. Web. 20 Oct. 2016.

Liu, Yuchen, Yayue Zeng, Li Liu, Chengle Zhuang, Xing Fu, Weiren Huang, and Zhiming Cai. "Synthesizing AND Gate Genetic Circuits Based on CRISPR-Cas9 for Identification of Bladder Cancer Cells." Nature Communications 5 (2014): 5393. Web.

Steigedal, Magnus, and Svein Valla. "The Acinetobacter Sp. ChnB Promoter Together with Its Cognate Positive Regulator ChnR Is an Attractive New Candidate for Metabolic Engineering Applications in Bacteria." Metabolic Engineering 10.2 (2008): 121-29. Web.

Valls, M. "Transient XylR Binding to the UAS of the Pseudomonas Putida 54 Promoter Pu Revealed with High Intensity UV Footprinting in Vivo." Nucleic Acids Research 31.23 (2003): 6926-934. Web.

         Adress:National Nanotechnology Research Center
                Bilkent University
                06800 Çankaya
                Ankara
                Turkey
         Phone:  +90 555 353 7220 
         Email:  unam-igem-2016@googlegroups.com