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Revision as of 03:40, 19 September 2015

Abstract

Specific detection of biomolecules is relevant in clinical diagnosis as well as toxicological, ecological or forensic analysis and many fields of research. However, individually developed antibodies are extremely expensive

Introduction

Method

We computationally designed a communication module for an HRP-F8 fusion DNAzyme using our software JAWS. Aptamers for ampicillin and ketamine were joined to the stem of the fused F8 DNAzyme to regulate its cleavage activity. The ketamine-binding aptamer was previously designed by our software MAWS to specifically target the anesthetic, while the ampicillin-specific aptamer was previously published Song2012. The fusion DNAzymes were tested against their respective ligand in solution, where 1 µM DNAzyme hemine solution was incubated together with the ligand, hydrogen peroxide, and TMB. Reactions were stopped using 1 mM hydrochloric acid.

Results

Ampicillin-reactive DNAzyme
Figure 1: Ampicillin-responsive HRP-F8 fusion DNAzyme.

A fusion of an ampicillin-binding aptamer and the HRP-F8 DNAzyme was incubated with hydrogen peroxide and TMP in either the presence or absence of ampicillin in A) reaction buffer or B) ampicillin-spiked energy drink.

Ketamine-reactive DNAzyme
Figure i+1: Ketamine-responsive HRP-F8 fusion DNAzyme.

Fusions of a ketamine-binding aptamer and the HRP-F8 fusion DNAzyme designed to switch either on or off in the presence of ketamine as well as negative controls (DNAzymes predicted by our software to not possess an efficient switching behavior) were incubated with hydrogen peroxide and TMP in either the presence or absence of ketamine.

Catalytic activity of the ampicillin-responsive DNAzyme drastically increased upon exposure to its ligand (Fig. 1A), indicated by the solution turning deep blue, while the negative control lacking ampicillin was only faintly coloured. To test this detection system in a more realistic setting, the DNAzyme was incubated in energy drink spiked with ampicillin. Here, a fainter change in color, presumably due to suboptimal buffer conditions was observed, with the ampicillin-spiked beverage still showing significant discoloration compared to the pure drink (Fig. 1B). From the two DNAzymes designed for ketamine one switched on in the presence of ketamine, while the other switched off. Both produced similar colors after the reaction was stopped (Fig 1 A+B). Thus, our software suite proved successfull in designing a detection system for a drug within less than a day of computation.