Team:SMTexas/Design

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Here is the list of genes we found to have detect various VOCs including ethanol, xylene, and formaldehyde.

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)

The frmR gene is induced by formaldehyde, a VOC that exists in higher concentrations in the breath of lung cancer patients. In E. Coli, frmR is followed by a promoter, which in turn is followed by an operator and the GFP coding gene. frmR, which functions as a regulatory gene, codes for a protein which acts as an active repressor for the operator. Thus, RNA polymerase cannot pass through the operator and transcribe GFP. Formaldehyde, which acts as a co-repressor, would bind to the protein and conform it into an inactive shape, allowing the RNA polymerase to pass through the operator and transcribe the GFP protein. Thus, if formaldehyde is present, the GFP gene is expressed, and the bacteria will glow in the presence of the VOC.

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 <h4> XylR Gene (Detects Xylene) </h4>
-The genetically-related expression of the XylR gene consists of many steps and parts, including promoters, regulator complexes, and proteins. The first part is the Pr promoter, which promotes the coding DNA sequence for the XylR gene itself. Following that, there is the ribosomal binding site portion of the sequence, which ensures that translation is initiated and that it occurs in the right place. Directly after this RBS is the exon itself, which naturally expresses the XylR protein, important to the E. Coli cell. Because this protein triggers a secondary response in the bacterium which is vital to Xylene detection, a double termination sequence is need to ensure that anything downstream of the XylR coding region is not expressed, potentially disrupting the reactions involved in the detection system. These stop codons, although short, are effective in terminating the expression of the sequence. The XylR protein that was created, given the that xylene is present in the bacterium, reacts with the VOC and becomes conformed. This newly conformed version of the protein can bind to the Pu promoter, which is part of an entirely different sequence. After a second ribosomal binding site (strong) is used after the promoter and YFP is placed directly after this RBS.1, the bacteria will grow green in the presence of 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.
 <table><tr><td width="1200" align="center"><img src="https://static.igem.org/mediawiki/2014hs/a/a5/XylR_Map.png"></td></tr></table>