Team:UFMG Brazil/Project/Parts

As a proxy for the detection of L-DNA, we decided to build first a generic DNA sensor that could be used to distinguish between the total amount of DNA on the feces of normal and diseased patients.

We wanted the reporter to be easily measurable on a lab, but also something that could be detected by a patient without any equipment. Thus, our choice was to use a split version of mChery Red Fluorescent Protein; mCherry’s red fluorescence can be quantified with ease on a fluorometer, and it can also be seen by the naked eye, since its emission fall on the visible spectrum. Its split version (which we call hemiCherrys A and B) work on a bimolecular complementation system [1] – that is, each of the two parts must be attached to other domains that can bring themselves to be close to each other, in a way that the function of the reporter is restored and allows for the fluorescence to be emitted after excitation. The separated parts do not emit fluorescence nor are able to restore complementarity by themselves.

To bring hemiCherrysA and B close to each other on a DNA molecule, we needed them to be attached to DNA-Binding domains that would bind close to each other on the substrate molecule, and also on a spread-manner fashion throughout the spectrum of DNA fragments. To accomplish that, we designed a TALE Domain using modified parts from the Freiburg 2012 collection [2] to bind to the GT (or CA) microsatellite – the most frequent non-single base repetitive DNA on the human genome [3]. Our designed TALE domain binds to a 12-base region containing six repetitions of the dinucleotide GT. Thus, on a longer DNA fragment spanning the microsatellite, two TALE domains can bind close to each other, each attached to a part of hemiCherry, restoring their functionality.

Of course, before this chimeric proteins can bind to the DNA on the feces, they must be secreted by the yeast or bacteria into the extracellular media. For that, we added a secretion signaling domain, according to the chassis: for gram-negative bacteria, we added the TorA twin-arginine translocase domain, a Type II-secretion system [4], wheras for yeast we used an exportation signaling domain from the Phosphatase-1 protein [5]. Two eleven-amino acid linker are used for avoiding allosteric interference between the three main domains. For a higher expression of the chimeric sensor, the coding region was inserted after a strong constitutive promoter, in conjunction with appropriate RBS and terminators.

References

[1]: Fan JY, Cui ZQ, Wei HP, Zhang ZP, Zhou YF, Wang YP, Zhang XE. Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein-protein interactions in living cells. Biochem Biophys Res Commun. 2008 Feb 29;367(1):47-53. Epub 2007 Dec 26. PubMed PMID: 18158915.

[2]: https://2012.igem.org/Team:Freiburg

[3]: Nadir E, Margalit H, Gallily T, Ben-Sasson SA. Microsatellite spreading in the human genome: evolutionary mechanisms and structural implications. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6470-5. PubMed PMID: 8692839; PubMed Central PMCID: PMC39047.

[4]: Thomas JD, Daniel RA, Errington J, Robinson C. Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli. Mol Microbiol. 2001 Jan;39(1):47-53. PubMed PMID: 11123687.

[5] Hashimoto Y, Koyabu N, Imoto T. Effects of signal sequences on the secretion of hen lysozyme by yeast: construction of four secretion cassette vectors. Protein Eng. 1998 Feb;11(2):75-7. PubMed PMID: 9605540.