"
Team:Lethbridge Canada/results
From 2013hs.igem.org
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 |
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 |
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
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 |
|---|---|---|
| 1 | 1KB DNA LADDER | 3µL |
| 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.
