Difference between revisions of "Part:BBa K1636000"

Revision as of 01:54, 19 September 2015

Lead-II-ions-specific aptamer

This aptameric sequence is capable of recognising and binding specifically to lead II ions. This sequence, which works as a single stranded nucleic acid molecule, is capable of forming a G-quadruplex by interacting with lead ions (Pb2+) while disrupting a partially complementary duplex.

Sequence and Features

The linker was ligated into the linearised plasmid pSB1C3 and previously cut with EcoRI and PstI, then a purification was performed (figure 1A). Several clones were selected to isolate plasmid by miniprep (figure 1). In order to analyse the integrity of plasmids an electrophoresis was run (figure 1B). Several plasmid isoforms were observed and the plasmids were suitable to be used as template in a PCR reaction to know the presence of the insert.

Figure 1. A) Scheme of aptamer cloning in pSB1C3 plasmid and B) electrophoresis with agarose gel at 0.8% to analyse plasmid extraction from different clones (lanes 2 and 3 with 10 microliters of each sample + 4 microliters of loading buffer).

To know if the aptamer was cloned in pSB1C3 a PCR was performed using universal primers VF2 and VR (figure 2A). Those primers amplify a PCR product of 299 pb when the insert is present (figure 1A). Plasmids from 2 different clones were tested and one of them had the PCR product of approximately 299 bp corresponding to the plasmid with insert (figure 2B, lane 3).

Figure 2. Analysis of the aptamer cloning into pSB1C3 plasmid by PCR. A) Plasmid scheme with the position of the cloned aptamer wich is flanked by the restriction sites EcoRI and PstI, also the alignment position of primers VF2 and VR is indicated. B) Plasmids from 2 different clones were used as templates for PCR with universal primers VF2 and V (lanes 2-3). Lane 1 contains Quick-Load® 2-Log DNA Ladder(0.1-10.0 kb), NEB.

Measurement of aptamer fluorescence by ZnPPIX and Pb(NO3)2

This essay was performed in order to check the ability that certain oligonucleotides have of capturing lead, including the aptamer (K1636000), the aptamer flanked by restriction enzymes sites EcoRI and PstI, scaffold 3 and DNAbot. The protocol used was based on the report of Li et al 2010.

Protocol based on Li et al, 2010 with several modifications described below: Reactions with 200 ul as final volume was prepared as following: - 6 uM of oligonucleotides 1 or 2 or 10 nM of oligonucleotides 3 or 4 - 10mM Tris-Ac buffer, 10 uM ZnPPIX - 50mM MOPS buffer, 10uM Pb(NO3)2.

Samples were transferred to a white opaque plate. Fluorescences were read at different times for 2 hours (time used in Li et al 2010) using a plate reader Synergy-H4 with an excitation wavelength of 420 nm and fluorescence emission at 600 nm. A set of tests were performed adding a previous temperature treatment for oligonucleotides consisting on keeping samples at 80°C for 10 min in a water bath, followed by slightly cooling down at room temperature and the addition of ZnPPIX, MOPS buffer and Pb(NO3)2. All reagents, except oligonucleotides 3 and 4, were in the same concentrations than the ones reported in the experiments of Li et al 2010.

Figure 3 depicts the fluorescence emission of oligonucleotides and some prepared blanks. We observed that Pb solution in nitric acid was a factor that contributed to the emission of fluorescence. We chose as final blank the reaction without Pb-HNO3 because DNA and ZnPPIX could emit fluorescence but lead would enhance that fluorescence emission. Moreover, this blank was also chosen since it was more stable than the others tested. Figure 3 shows that absence of Pb solution changed dramatically the fluorescence emission. This blank was used to correct the RFU of reactions, Figures 4 and 5 depict corrected RFU of reactions.