Team:Lethbridge Canada/results


Silver Stained Tris-Tricine PAGE

Lane Contents Volume
1 Protein Molecular Weight Marker 20μL
2 Cbf5-Nop10-Gar1 20μL
3 Buffer A wash 30μL
4 Buffer B wash 30μL
5 Buffer E Wash 30μL
6 S-30 s/N 30μL
7 Buffer E – Concentrated Preprooxyphysin 30μL
Gel Gel

To detect our prepro-oxyphysin proteins we did a Silver Stain 16.5% Tris-Tricine PAGE. The reason for doing a Silver Stain is that it is 10x more sensitive than the standard Coomassie stain. The tricine in the Tris-TRicine PAGE allows for better and more distinct separation of small proteins in the gel. In Lane 2, we did a control of Cbf5-Nop10-Gar1 proteins. Cbf5 is 43 kDa, Gar1 is 12.3 kDa and Nop10 is only 7.2 kDa. Nop 10 is outlined in red. Prepro-oxyphysin is slightly larger than Nop10 at 10kDa. The bands we suspect are prepro-oxyphysin are outlined by orange. They lowest visible marker on the protein ladder is 14 kDA.

Anti-His Slot Blot

Lane # Contents
Lane 1: 100μL Oxytocin in 200μL TBS
Lane 2: 200μL Oxytocin in 100μL TBS
Lane 3: 250μL Oxytocin in 50μL TBS
Lane 4: 300μL Oxytocin
Lane 5: 25pM TruB-His positive control
Slot Blot confirming His-Tag

Below is a Slot Blot done to confirm the presence of a Oxytocin-Neurophysin I. During the synthesis of our gene, a histidine tag was added following the protein. A Slot Blot test works by utilizing two types of antibodies, and will chemiluminesce if our protein is present. The primary antibody is sensitive to histidine and accordingly, will bind to it. The secondary anitbody is sensitive to the primary antibody, but has a chemiluminescent enzyme attached to it. Should the secondary antibody bind to the primary antibody, they will both remain attached to the histidine tag on the protein and when induced with luminol, p-coumaric acid and hydrogen peroxide, will glow. Accordingly, we can detect this chemiluminescence and confirm the presence of our protein.

We used a 25pM sample of the protein TruB with a histidine tag which is seen in lane 5. In lanes 1,2,3 and 4, faint bands are seen on the slot blot. This is indicative of the histidine tag attached to Oxytocin-Neurophysin I.

1%Agarose Gel of K314100_OXT

Final Gel Oxytocin Conformation-Negative/Enhanced
Final Gel Oxytocin Conformation-Original

We have shown that we have successfully assembled the Oxytocin-Neurophysin I gene, complete with signal sequence, into pSB1C3. The expected size of the Oxytocin-Neurophysin I gene is 406 base pairs. The expected size of K314100_Oxytocin-Neurophysin I is 924 base pairs. The expected size of K314100_Oxytocin-Neurophysin I_pSB1C3 is 2994 base pairs. The Oxytocin-Neurophysin I gene is clearly seen in lanes 5, 8, 10 and 12.

Lane # Contents Volume
2 K314100_pSB1C3 cut EcoRI 15µL
3 K314100_pSB1C3 cut EcoRI and PstI 15µL
4 Oxytocin-Neurophysin I_PUC57a cut EcoRI 15µL
5 Oxytocin-Neurophysin I_PUC57a cut EcoRI and PstI 15µL
6 Gel Extraction of pSB1C3 cut EcoRI and PstI 15µL
7 K314100_Oxytocin_Neurophysin I cut EcoRI 15µL
8 K314100_Oxytocin_Neurophysin I cut EcoRI and PstI 15µL
9 K314100_Oxytocin_Neurophysin I cut EcoRI 15µL
10 K314100_Oxytocin_Neurophysin I cut EcoRI and PstI 15µL
11 K314100_Oxytocin_Neurophysin I cut EcoRI 15µL
12 K314100_Oxytocin_Neurophysin I cut EcoRI and PstI 15µL

Sequencing results for K314100_OXT

Future Directions

Next year, we hope to continue this project where we left off. We will further characterize our Oxytocin-Neurophysin I construct. We will also complete assembly of the NEC I construct and begin to characterize it as well. We will also continue to optimize the NEC I construct by investigation promoter and RBS efficiency.