Difference between revisions of "Part:BBa K2921250"

Latest revision as of 09:25, 21 October 2019

Promoter + RBS + 6xHIS + OpdA + GS-linker + amilCP + Double Terminator

This construct constitutively expresses OpdA (Basic part: BBa_K215090) linked with amilCP (Basic part: BBa_K592009). According to iGEM09_Washington’s basic part page of BBa_K215090, OpdA is a phosphotriesterase from Agrobacterium radiobacter that can degrade organophosphate pesticides.The amilCP serves as a functional color reporter, allowing for convenient visible detection of the location of OpdA.

Construct Design

This construct was created to constitutively express OpdA-GS-amilCP. Sequences used for the promoter, RBS, and double terminator came from parts included in the iGEM distribution kit. This construct consists of a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, the protein-coding gene OpdA (Basic part: BBa_K215090), and a double terminator (BBa_B0015) to end transcription. In addition, for downstream applications, we included a hexahistidine tag (6xHis) after the RBS and prior to the OpdA ORF for easy protein purification.To ensure that the binding protein and colored protein were fused, we inserted a flexible glycine-serine linker between the binding protein and the chromoprotein.

This entire construct was synthesized by Twist Bioscience.

PCR Check Results

The part was confirmed by PCR using the primers VF2 and VR, as well as sequencing by Tri-I Biotech.

We confirmed the size of K2921250 using the primers VF2 and VR, which resulted in the expected size of around 1.9kb.

Characterization

We used SDS-PAGE to check for OpdA-GS-amilCP expression in E. coli carrying our construct. Bacterial cultures expressing either OpdA-GS-amilCP or BBa_K880005 (empty vector) were grown overnight at 37°C, lysed and run on SDS-PAGE gels. OpdA-GS-amilCP is approximately 63 kDa, and we observed a strong signal at that size in the OpdA-GS-amilCP lysate sample which was not present in the empty vector sample, suggesting that OpdA-GS-amilCP is being expressed in the transformed E. coli.

E. coli carrying BBa_K2921250 was lysed and run through a nickel column (GE Healthcare, 11-0033-99) which bound our His-tagged OpdA-GS-amilCP protein. Bound proteins were eluted with elution buffer and the purified proteins were subject to SDS-PAGE to check the size. The expected size of OpdA-GS-amilCP is approximately 63 kDa, but we observed a strong signal at approximately 75 kDa in the OpdA-GS-amilCP lysate sample, which was not present in the empty vector sample. This discrepancy in size is likely due to post-translational modifications on the protein such as phosphorylation and glycosylation. Similarly, we observed a signal at 75 kDa in the purified protein sample, suggesting that we were able to successfully purify it.

The left gel and right gel are each individual gels. To verify OpdA-GS-amilCP expression in E. coli, we subjected OpdA-GS-amilCP lysate (left gel) and OpdA-GS-amilCP purified protein (right gel) to SDS-PAGE, expecting a signal at around 63 kDA. On the left gel, we saw a signal at around 75 kDa in the OpdA-GS-amilCP lane, but not in the empty lane that was used as a control. This discrepancy in size is likely due to post-translational modifications on the protein such as phosphorylation and glycosylation. On the right gel, we saw a similar signal at around 75 kDa in the OpdA-GS-amilCP purified lane.

Sequence and Features