siRNA confirmation

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Sequencing

It is fundamental for us to make sure that all our BioBricks have the correct sequence before sending them to Boston for the parts registry. Therefore, we sequenced every single piece, to confirm that the ligated products into the pSB1C3 backbone correspond to the original sequence.

The bio bricks cloned by the IGEM TEC CEM 2017 team in PSB1C3 were sequenced in order to test if the inserts for the siRNAs and T7 terminator were really ligated. For the following samples of BSLA devices (Blue chromoprotein, forward siRNA, loop and reverse siRNA): 1, 2, 3, 4 and 5 the sequencing was made using the VF2 primer, although it was possible to confirm the presence of the blue chromoprotein, the reaction couldn´t show us the presence of the siRNAs constructs because of the length of the protein gene before them. In these cases, a reaction using the VR primer is recommended since there are approximately in each case a total of 220-230 pb since the reverse primer to the siRNAs constructs. The single presence of the siRNAs sequence (5’-3’) in PSCB1C3 without all the machinery (blue chromoprotein, loop and siRNA anti sense) was confirmed for the RacI and SOD constructs. The Wnt construct wasn’t sequenced and the results for the Awd construct weren´t conclusive. Also, the presence of the sequenced used as the control of the siRNAs (CNT) was confirmed to be cloned in PSB1C3 (sample 7). The sample 8 correspond to the single sequence of the T7-Terminator used during the construction of the BSLA devices, it was confirmed to be cloned in the PSB1C3 For the samples of BSLA device, we have to repeat the sequencing using the VR primers for the BSLA-SOD and BSLA-RACI, so for this samples we have the sequence Forward and reverse with the objective to performed to seek the siRNA construction before the blue chromoprotein, however the results weren´t conclusive since the alignment showed the correct base pairs just after the reverse primer but not the same sequence before the terminator. And finally the sequencing for BSLA-AWD in PSB1C3 wasn’t repeated, since it was given to the team by the Gene script sponsorship, and once cloned in PSB1C3 the colonies became blue. *All the samples were sequenced in a ABI PRISM 310 equipment

Blue BSLA colonies

The following figures show blue flouresce colonies of E.coli Ht115, the color of the bacteria represents the presence of the BSLA construct therefore production of siRNA.

Real Time PCR