Team:TCU Taiwan/Results
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To get the SgrS binding site sequence we need, we use the designed primers to run the PCR with the E. coli BL21 whole genomic DNA. The primers that we designed are expected to produce the product with 255 bp, and the result that we got after the experiment through the gel electrophoresis, the band of the expected product was located between 200~300 bp.(See picture1) | |||||||||||
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picture2 |
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Then we use the SgrS binding site sequence and the red chromoprotein (BBa_K592012) sequence together with the other pairs of the designed primers. For this segment, the expected product should be 936 bp. After the confirmation through the gel electrophoresis, we can make sure that the band of the product is located around 900 bp, it shows that we have fused the protein successfully.(See picture2) | |||||||||||
| Then we load our samples into the Gel to run the gel electrophoresis. Our Samples are SgrS binding side into PSB1C3, the fusion protein between SgrS and Red color chromoprotein into PSB1C3 and the fusion protein between SgrS and Red color chromoprotein into PQE60. Next, we transform the plasmid PSB1C3 into the DH and PQE60 into BL21.(see the picture3) | |||||||||||
picture3
After the transformation using spread plate method, we begin to select the colony after 14 hours of transformation, then we extract the plasmid from what we have already transformed. | When the Plasmid already extracted, we start to run the PCR by using designed primer, to determine that we really got the desire sequence we run the gel electrophoresis.(see the picture4)
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picture4
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picture5&picture6
To confirmed our PCR really work or not, we did once again PCR. This also wants to prove plasmid extraction whether we really got the plasmid that we want to extract or not. this confirmation also pointed to confirmed SgrS binding site linked with PSB1C3. | And this confirmation also pointed to confirmed the fusion construct between SgrS and Red color chromoprotein linked with pQE60.(see the picture5&picture6) Base on the above result we also confirmed is that our inserted sequence presence or linked with the chosen vector. After it was confirmed, we begin to transform our ligation product into the E.coli. |
Conclusion
In this time we want produce the ligation product which is linked with the plasmid. Right now in the Experiment, we discover some problem. | In the beginning, we expect that when we ligase the SgrS binding site with red color chromoprotein and transform it into the E.coli BL21 we can see the red color from the colony of the transformed BL21. Unfortunately, because the system of the SgrS and the media that we used to grow the colony of transformed E.coli, Our product start to react. When SgrS binds to the mRNA SgrS binding site, and the binding of this complex will hydrolyze our mRNA red color chromoprotein resulting no red color produce. Therefore, by using the result that we have already mentioned, we can confirm that our SgrS system is in the good shape. To make a 100% confirmation about our product, we decide to take the ligation product and do the sequencing method to confirm whether this the right sequence or not. | |||||
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