Part:BBa_K3725022:Design

Improved Fusarium Toehold w/ GFP Reporter
Design
The construction of a disease-specific biosensor required us to find a gene unique to the pathogen. When the switch turns on and GFP is expressed, we can confirm that the specific pathogen is present. For the detection of Fusarium oxysporum f. sp. lycopersici, Lambert iGEM focused on the FRP1 gene. This gene was selected because it was required for pathogenicity and was unique to the species of interest. Biosafety note, the trigger sequence is not the full transcript sequence and therefore poses limited biosafety. We obtained the sequence via UniProt, an online database of protein sequences. Lambert iGEM used the code from Takahashi et. al provided by Megan McSweeney from the Styczynski Lab at the Georgia Institute of Technology to design the switch and trigger sequences on NUPACK. The team selected the pair from NUPACK with the lowest normalized ensemble defect (NED) to maximize the chances of successful compatibility. Once we obtained the sequences for the toehold pair, we constructed the toehold and trigger via SnapGene.

This part is different from Part BBa_K3725020, the Fusarium Toehold w/ GFP reporter, in that there are differences in the sequence that change the hairpin loop structure of the toehold switch. This was the pair with the second-lowest normalized ensemble defect from NUPACK. As compared to the Fusarium Toehold w/ GFP reporter, the free energy of the hairpin loop of our redesigned part is higher, at -25.30 kcal/mol versus -26.10 kcal/mol.

Figure 1: BBa_K3725022 - Improved Fusarium Toehold w/ GFP Reporter Construct

Assembly Compatibility:

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

  Illegal NheI site found at 861
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 777
  

Design Notes

design considerations

Source

source of this part