Part:BBa_K2921570

Promoter + RBS + Metallothionein + EAAAK-linker + mRFP + Double Terminator

This gene codes for a colored metal-binding fusion protein: Metallothionein (Composite part: BBa_K1460002) linked with mRFP (Basic part: BBa_E1010). According to iGEM14_Cornell’s composite part page of BBa_K1460002, Metallothionein is a metal-binding protein that can tightly chelate heavy metal ions by forming a strong coordination bond. The mRFP serves as a functional color reporter, allowing for the convenient visible or UV detection of the location of Metallothionein.

Construct design

This construct was created to constitutively express Met-mRFP fusion proteins. 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 Metallothionein (Composite part: BBa_K1460002), the chromoprotein gene mRFP (Basic part: BBa_E1010), 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 Metallothionein ORF for easy protein purification. To ensure that the binding protein and colored protein were fused, we inserted a rigid EAAAK-linker between the binding protein and the chromoprotein.

This entire construct was synthesized by Twist Bioscience.

PCR

PCR check results, using the primers VF2 and VR, and sequencing results by Tri-I Biotech confirm this construct.

We confirmed the size of K2921570 using the primers VF2 and VR, which resulted in the expected size of around 1.4 kb.

Characterization

We used SDS-PAGE to check for Met-EAAAK-mRFP expression in E. coli carrying our construct. Bacterial cultures expressing either Met-EAAAK-mRFP or BBa_K880005 (empty vector) were grown overnight at 37°C, lysed and run on SDS-PAGE gels. The expected size of Met-EAAAK-mRFP is approximately 40 kDa, but we observed a strong signal at approximately 48 kDa in the Met-EAAAK-mRFP 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.

To verify Met-EAAAK-mRFP expression in E. coli, we subjected Met-EAAAK-mRFP lysate to SDS-PAGE, expecting a signal at around 40 kDa. Instead, we saw a signal at around 48 kDa in the Met-EAAAK-mRFP 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.

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 789
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1614
  Illegal AgeI site found at 1726
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 201