aldB Gene (Detects Ethanol)

The aldB gene codes for a functional aldehyde dehydrogenase, which is directly induced by ethanol. Metabolism of the alcohol consists of its transformation into an aldehyde and then into a carboxylic acid in activating several related pathways. The acid reacts with the BarA histidine sensory kinase, a signalling enzyme involved in a two component signal transduction system present in E. coli, to catalyze the breakdown of various carboxylic acids. The kinase is additionally responsible for the induction of RpoS, a regulatory gene of aldB that directly opposes fis during the activation of the aldB operon. Eventually, BarA triggers a series of vital reactions that affect the Crp-cAMP regulatory mechanism, a dual complex that controls the expression of the aldB coding sequence. In the complex, cAMP conforms the shape of Crp, also known as CAP (catabolite activator protein). This newly conformed Crp then attaches to the promoter and contributes to the initiation of transcription of the aldB operon. Downstream of aldB, CFP (cyan fluorescent protein) is expressed and the bacteria exhibits cyan fluorescence.

XylR Gene (Detects Xylene)

The genetically-related expression of the XylR gene consists of promoters, regulator complexes, and proteins that all aid in the expression of fluorescent proteins. The initial DNA sequence Pr promotes the expression of XylR gene exon itself. Shortly after, it is followed by a ribosomal binding site that orchestrates the timing and efficiency of translation. The naturally expressing XylR sequence succeeds the RBS and undergoes strict regulation of the Pr promoter. Because this protein triggers a secondary response in the bacterium which is vital to Xylene detection, a double termination sequence is essential to the discontinuation of sequences downstream of the XylR coding region is not expressed which can disrupt the reactions involved in the detection system. These stop codons, which are short and effective, operate with a stem-loop that possesses both forward and reverse termination mechanisms. The expressed XylR protein then reacts with xylene and is conformed to accommodate a secondary gene sequence. This newly conformed version of the protein can then bind to the Pu promoter. After a second ribosomal binding site (strong) is subsequently intiated and YFP is expressed, which supersedes the RBS, the bacteria will exhibit yellow fluorescence to indicate a positive test.

frmR Gene (Detects Formaldehyde)

Formaldehyde induces the frmR gene, functioning as a regulatory gene of Green Fluorescent protein. When transcribed, it expresses a regulatory protein that binds to an downstream operator that prevents the movement of RNA polymerase. Under typical conditions, the promoter downstream of the regulatory gene increases the affinity of RNA polymerase to the DNA strand, but the transcription enzyme cannot bypass the operator and transcribe GFP, the gene that is ultimately under regulation. In such a scenario, the regulatory protein that frmR expresses functions as a repressor and effectively inhibits transcription of the coding sequence. Formaldehyde, on the other hand, induces the transcription of GFP and ultimately causes bacterial fluorescence. Acting as a corepressor, the VOC binds to the regulatory protein and conforms it into an inactive shape, allowing for the passage of RNA polymerase through the operator and transcribe the GFP protein.

General:

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American Lung Association. (2012, April). Providing Guidance on Lung Cancer Screening To Patients and Physicians. Retrieved from http://www.lung.org/lung-disease/lung-cancer/lung-cancer-screening-guidelines/lung-cancer-screening.pdf

Pfizer Oncology. Lung Cancer and Biomarkers. Retrieved from http://www.lungcancerprofiles.com/lung_cancer_and_biomarkers.

Hakim, M., Broza, Y. Y., Barash, O., Peled, N., Phillips, M., Amann, A., & Haick, H. (2012). Volatile Organic Compounds of Lung Cancer and Possible Biochemical Pathways. Chemical Reviews, 112(11), 5949-5966. doi: 10.1021/cr300174a

Fu, X., Li, M., Knipp, R. J., Nantz, M. H., & Bousamra, M. (2013). Noninvasive detection of lung cancer using exhaled breath. Cancer Medicine, 3, 174-181. doi:10.1002/cam4.162

Mazzone, MD, MPH, FRCPC, FCCP, (July 2008). Analysis of Volatile Organic Compounds in the Exhaled Breath for the Diagnosis of Lung Cancer. Journal of Thoracic Oncology . 3 (7), pp.774-781

Phillips, M., Gleeson, K., B Hughes, J. M., Greenberg, J., Cataneo, R. N., & Baker, L. Volatile organic compounds in breath as markers of lung cancer: a cross-sectional study . Early Report, 353, 1930-1936.

Fuchs, P., Loeseken, C., Schubert, J. K., & Miekisch, W. Breath gas aldehydes as biomarkers of lung cancer. International Journal of Cancer, 126, 2663-2670.

Bajtarevic, A., Ager, C., Pienz, M., Klieber, M., Schwarz, K., Ligor, M., et al. Noninvasive detection of lung cancer by analysis of exhaled breath. BMC Cancer, 9.

Wang, Y., Hu, Y., Wang, Di., Yu, K., Wang, L., Zou, Y., Zhao, C., Zhang, X., Wang, P., & Ying, K. The analysis of volatile organic compounds biomarkers for lung cancer in exhaled breath, tissues and cell lines. Cancer Biomarkers, 11, 128-137. doi:10.3233/CBM-2012-00270.

Clinical Trials. (2012). Exhaled Breath Biomarkers in Lung Cancer. Retrieved from http://clinicaltrials.gov/show/NCT01386203

xylR gene:

EcoCyc (n.d. )Escherichia coli K-12 substr. MG1655 Polypeptide: XylR transcriptional activator. Retrieved from http://ecocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=EG20253-MONOMER

Nucleic Acids Research 39:D583-90 2011

WikiGenes (n.d.) xyIR-xyIR Pseudomonas Putida. Retrieved from http://www.wikigenes.org/e/gene/e/1218757.html SmoColi. (n.d.) How we calculated the concentration of m-xylene in the medium…. Retrieved from https://2011.igem.org/Team:ETH_Zurich/xylene

aldB gene:

Xu, J., Diderichsen, B., Ho, K. K. Wiki Genes. aldB - aldehyde dehydrogenase BEscherichia coli str. K-12 substr. MG1655. Retrieved From http://www.wikigenes.org/e/gene/e/948104.html

Xu, J., & Johnson, R. C. National Center for Biotechnology Information. aldB, an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp. Retrieved From http://www.ncbi.nlm.nih.gov/pmc/articles/PMC177007/

EcoCyc. (n.d.). Escherichia coli K-12 substr. MG1655 Enzyme: acetaldehyde dehydrogenase. Retrieved From http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG12292

PortEco. (2013). aldB:gene. Retrieved from http://ecoliwiki.net/colipedia/index.php/aldB:Gene

frmR gene:

Tokyo Metropolitan University. (n.d.) Parts. Retrieved from https://2012.igem.org/Team:TMU-Tokyo/Parts

PortEco. (n.d.) Retrieved from http://heptamer.tamu.edu/fgb2/gbrowse/MG1655/?plugin=FastaDumper&q=NC_000913:378830..379105&plugin_action=Go

PortEco. (2013). frmR gene. Retrieved from http://ecoliwiki.net/colipedia/index.php/frmR:Gene Wang, S., Deng, K., Zaremba, S., Deng, X., Lin, C., Wang, Q., et al. (2009, August 7). Transcriptomic Response of Escherichia coli O157:H7 to Oxidative Stress. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2753066/ EcoCyc. (n.d.) Escherichia coli K-12 substr. MG1655 Polypeptide: regulator protein. Retrieved from http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=G6209