Difference between revisions of "Part:BBa K1614009"

Revision as of 08:11, 18 October 2019

software generated kanamycin aptamer (candidate 1)

Aptamer binding kanamycin created by the MAWS (http://2015.igem.org/Team:Heidelberg/software/maws) Software from iGEM Team Heidelberg, further validated by an HRP based assay (http://2015.igem.org/Team:Heidelberg/project/hlpd).

Usage and Biology

1. Our vision & The parts we characterize

Team DUT_China_A 2019 dedicate themselves to convey the concept of functional DNA application across iGEM and we would like to provide iGEM conmunity with available functional DNA. Therefore, we add experimental characterization data to the aptamers constructed by Team Heidelberg and we improve their parts.
They conceived and implemented an open-source software for the design of aptamers (MAWS) as a fast and affordable alternative to the laborious SELEX procedure.
1.BBa_K1614009- software generated kanamycin aptamer (candidate 1)
2.BBa_K1614013- software generated kanamycin aptamer (candidate 2)
3.BBa_K1614017- software generated kanamycin aptamer (candidate 3)
To test these aptamers whether they can bind to kanamycin, we put forward a plan, Gold nanoparticle-based colorimetric detection.

2. Principle

Gold nanoparticles (Au NPs) covered by Citrate ion are charged negatively and they repel each other, remaining dispersive in the solution. After adding NaCl into this solution system, the double electrode layer of the Au NPs are destroyed and they tend to being coagulated, resulting the color of the solution changes from red to blue-purple, and the spectrum of the solution shifts red. The absorbance of 620nm obviously increase. However, short ssDNAs are readily adsorbed onto the surface of AuNPs without any modification and that such ssDNA-treated AuNPs are more stable than untreated ones. Adding aptamers into the system, the citrate root of nano gold surface is replaced. AuNPs can still remain negetively charged instead of being coagulated even if there is NaCl due to the non-specific interaction between DNA side chain phosphate groups and Au NPs, keeping the solution color red. When kanamycin, the determinand is added in to the solution, the adaptor configuration changes and falls off the surface of the Au NPs because there is specific affinity between the kanamycn and the aptamers. AuNPs is going to coagulate. The changing concentrations of kanamycin lead to the changing AuNPs solution spectra. Adopting this method, the specific binding of the aptamer to kanamycin can be tested and the kanamycin in the solution can be easily and quickly detected.

3. Materials & Protocol

3.1 Materials

Kamanycin aptamers
① 5‘-TTCTCTC-3’
② 5‘-CGGGGAT-3’
③ 5’-GCTGTCG-3’
Tris-buffer（10mM Tris-HCl，50mM NaCl，5mM KCl，5mM MgCl2，PH=7.0）

3.2 Protocol, Results and Discussion

3.2.0 Preparation for Au NPs
Add 125mL HAuCl(1mM) water solution to 250mL round bottom flask. Heat it until boils then quickly add 12.5 mL (38.8mM) sodium citrate solution into this system. Heat for 10min, remove the heating sleeve, stir for 15min. Cool to room temperature and obtain Au NPs with particle size of about 13nm.

3.2.1 1.Exploration on the least concentration of NaCl to precipitate a certain concentration of Au NPs
We fabricate Au NPs systems with different concentration of NaCl in 96 well plates and measure their absorbance ratios at wavelengths of 520 nm and 620 nm, which is shown in figure.1 and figure.2.

Figure.1 A620/A520 of Au NPs systems determined by the concentration of NaCl.
The concentration of gold nanoparticle is 150 nM.

Sequence and Features