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.

Figure 3. Blue fluorescence observed in colonies of E. coli HT115, indicating the presence of our BSLA construct.

Figure 4. Blue fluorescence observed in colonies of E. coli HT115, indicating the presence of our BSLA construct.

Real Time PCR

Since the focus of this project is to verify the effective gene silencing with the four different siRNA we designed, the direct measurement of the amount of mRNA in the cells was required. The first step was to perform a Two-Step Reverse Transcriptase PCR to find out where do the siRNA molecules hybridize with the mRNA. The results are shown below. .

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Two-Step Quantitative Reverse Transcriptase PCR

Table 1. Expected products for the amplifications in the Two-step RT-PCR.

Product	Expected size
Tubulin	195 bp
Rac I	193 bp
WNT	200 bp
AWD	180 bp
SOD	199 bp

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Diaphorina citri primary cell culture

The main objetive was to develop a primary culture of Diaphorina citri cells, to be transfected with our siRNA and analyzed through flow cytometry. The transfected siRNA sequences were tagged with Alexafluor so they would be visible. This protocol was repeated several times under different conditions, and different compositions of the culture medium were used, which can be seen below. The cells that were cultured in Medium A were able to survive after twelve hours, and replicated at a slow rate. Because of this, the flow cytometry was not performed.

Reagent	Medium A	Medium B
200 mM L-Glutamine	1 mL	1 mL
Penicillin and Streptomycin 1000X	1.5 mL	1.5 mL
Fungizone 500X	0.2 mL	0.2 mL
Schneider Medium	72 mL	80 mL
199 Medium 10X with Hanks’ salts	6 mL	-----
Fetal Bovine Serum (heat-inactivated)	20 mL	20 mL
0.06 M Histidine solution	1 mL	-----

Figure 1. Cell culture 12 hours after extraction.

Figure 2. Cell culture 2 days after extraction, observed under an inverted microscope at 10x. No significant cell replication can be observed compared to the first day,

Figure 3. Cell culture 2 weeks after extraction, observed under an inverted microscope at 10x. Cell replication can be observed.

Diaphorina citri laboratory development

Special cages were built for rearing and reproduction of Diaphorina citri specimens across three months. Each cage was double lined with fine white mesh to prevent the insects from escaping while allowing us to inspect them. On top, a set of bulbs was placed and connected to a timer, to control daily light hours, which were set at 16 hours of light and 8 of darkness. Additionally, two fan heaters were placed at a distance of 1 meter, which increased the temperature inside the cages to up to 30ºC. While this is not the ideal temperature for reproduction, the presence of eggs and nymphs could be observed after two weeks.

The initial population across all cages was of 80 insects, and after approximately one month an increase of almost double was observed. However, due to the psyllid's lifespan, a rapid decline was observed shortly after. In addition to this, the increase in population made it harder for each psyllid to feed on one plant, which increased mortality.

Figure 1. Detail of mesh lining the interior of the cages. The size of the mesh prevents the insects from escaping while providing light, air flow and visibility.