Part:BBa_K2921575

Metallothionein + EAAAK-linker + mRFP

This gene codes for colored metal-binding fusion protein: Metallothionein (Composite part: BBa_K1460002) linked with mRFP (Basic part: BBa_E1010). According to iGEM14_Cornell’s basic 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

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 part is a subunit of the larger construct K2921570 which is identical to K2921575 but includes a promoter, RBS, and terminator. The following PCR check results and characterization is based on K2921570.

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.

In this gel, we ran the construct K2921570: Strong Promoter-RBS + 6xHis + Metallothionein(CRS5) + EAAK-linker + mRFP + Double Terminator, which contains the basic part K2921575. 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
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 706
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1531
  Illegal AgeI site found at 1643
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 118