Part:BBa_K2921450

Promoter + RBS + 6xHIS + MBP + GS-linker + mRFP + Double Terminator

This construct constitutively expresses a colored metal-binding fusion protein MBP (Basic part: BBa_K346004) linked with mRFP. According to iGEM10_Peking’s composite part page of BBa_E1010, MBP is a lead binding peptide that can accumulate lead ions from aquatic environment. The mRFP serves as a functional color reporter, allowing for the convenient visible or UV detection of the location of MBP.

Construct design

This construct was created to constitutively express MBP-mRFP. 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 MBP-mRFP (Basic part: BBa_K346004), and a double terminator (BBa_B0015) to end transcription. 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. In addition, for downstream applications, we included a hexahistidine tag (6xHis) after the RBS and prior to the MBP ORF for easy protein purification.

This entire construct was synthesized by Twist Bioscience.

PCR

The part was confirmed by PCR using the primers VF2 and VR

We confirmed the size of K2921450 using the primers VF2 and VR, which resulted in the expected size of around 1.2 kb.

Characterization We used SDS-PAGE to check for MBP-GS-mRFP expression in E. coli carrying our construct. Bacterial cultures expressing MBP-GS-mRFP were grown overnight at 37°C, lysed and run on SDS-PAGE gels. The expected size of MBP-GS-mRFP is approximately 43 kDa, but we did not see a signal at the right size in the MBP-GS-mRFP lane. We sequenced our synthesized construct and discovered the sequence was wrong. Therefore, we did not perform any functional tests on this construct.

To verify MBP-GS-mRFP expression in E. coli, we subjected MBP-GS-mRFP lysate to SDS-PAGE, expecting a signal at around 43 kDa. However, we did not see a strong signal at the right size in the MBP-GS-mRFP lane so we sequenced our synthesized construct and discovered the sequence was incorrect. Hence, we did not perform any functional tests on this construct.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1158
  Illegal AgeI site found at 1270
  
• 1000
  COMPATIBLE WITH RFC[1000]