We did a real time PCR, that demonstrates the mRNA expression vs the amounts of siRNA. The theoretical values are demonstrated as T-Xm and the values obtained in the experiment are represented as Xm. The d and θ were adjusted, .005 and 29.1 respectively, the obtained results prove that the transfection efficiency is of 42.9%. The maximum degradation rate observed as d resulted to be even lower than expected, the first value was .001.

The following table shows the values for each point using the equation.

Table 1. Xm and T-Xm values.

The following graph shows that the mRNA degradation does exist, but it doesn’t have the same behavior it is not as reduced as the mode, this means that at a bigger concentration the greater the degradation induced by the siRNA.

siRNA confirmation

The siRNA confirmation protocol was made to test, with the real time PCR, the presence of the siRNAs designed by the iGEM Tec Cem team. The four devices capable of producing the siRNAs were tested: BSLA-SOD, BSLA-WNT, BSLA-RACI and BSLA -AWD. The protocol used for this purpose is based on the liquid northern hybridization proposed by Wang X., Tong Y. and Wang S. (2010). The protocol is described as a novel form to detect miRNAs. We expect to detect the siRNAs that our different constructs can produce taking advantage of their hybridization with the forward and reverse templates of their DNA sequences and the ability of the exonuclease to digest the non-hybridized RNA sequences.

First BSLA siRNA confirmation

Second BSLA siRNA confirmation

The first test of the control (only RNA of HTT115) revealed the presence of two bands in the no-denaturing gel when no exonuclease is added to the reaction of approximately 150 pb and 100 pb respectively. When exonuclease is added to the same sample a little band is observed below the 50 bp. When 4 μL of a 100μM dna oligo was added to the same sample, the samples seem to migrate better than the last two and a great amount of material stayed in the low part of the gel, probably because of the high amount of non-hybridized oligo.

For the BSLA samples, interestingly, the behavior between the samples treated with the forward and reverse DNA template was different. The RNA samples treated with the forward template sequence migrated less than the ones treated with the reverse, probably because they really hybridized with the siRNA. The RNA samples treated with the reverse sequence migrated really similar to the RNA of HT115 treated with the oligo and exonuclease, a behavior that might mean that the oligos didn´t hybridize.

We expected to observe hybridization with both templates (forward and reverse), since the construction for each siRNA has their own sense and anti-sense sequence. The results in both confirmations might mean that the siRNA might hybridize with the forward sequence but no with the reverse. Together with the real time PCR we can conclude that we have a certain amount of siRNA but to characterize their hybridization, we should changes the amount of material for the experiment.

The second confirmation of siRNA was repeated with less RNA material (2 μg instead of 5 μg) and more units of exonuclease (20 instead of 10) expecting to distinguish more defined bands. Nevertheless, since it was used a lower dilution of GelRed seems brighter than the first confirmation and the observed material is similar. Next studies might use less RNA and oligos as in the protocol proposed by Wang X., Tong Y. and Wang S. (2010) where they used 1 μg of RNA.

Blue BSLA colonies

The following figures show blue fluorescent colonies of E. coli HT115. The color of the bacteria represents the presence of the BSLA construct and production of siRNA.