Team:Lambert GA/Design

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DESIGN

PARTS DESIGN

Megan McSweeney from the Styczynski Lab at the Georgia Institute of Technology provided us with Takahashi et. al's Nucleic Acid Package (NUPACK) software design code, allowing for our team to design toehold switches and complementary triggers for the pathogens of interest: Fusarium and Phytophthora. The team selected the toehold-trigger pairs 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. The switch part is composed of eight random base pairs to prevent our prefix and suffix from being compromised due to degradation, the BioBrick prefix and suffix, the T7 promoter (BBa_J64997), the switch sequence, the GFP reporter (BBa_E0040), and the T7 terminator (BBa_K731721). Sharing similarities with the switch sequences, the trigger is also composed of eight random base pairs, the Biobrick prefix and suffix, the T7 promoter (BBa_J64997), trigger sequence and the T7 terminator (BBa_K731721). We utilized the Anderson T7 promoter (BBa_J64997) to produce a strong expression of the downstream reporter protein, GFP. Lambert iGEM then ordered the parts from Integrated DNA Technologies and began the cloning process.

Figure 1. NUPACK’s calculated optimal toehold switch structure designs for the Fusarium (left) and Phytophthora (right) toeholds.

Figure 2. BBa_K3725020: Fusarium Toehold w/ GFP Reporter Construct

The Fusarium Toehold w/ GFP Reporter (BBa_K3725020) is composed of four basic parts: the T7 promoter (BBa_J64997), the switch sequence (BBa_K3725050), a GFP reporter (BBa_E0040), and the T7 terminator (BBa_K731721).

Figure 3. BBa_K3725070: T7 Fusarium Trigger Construct

The T7 Fusarium Trigger (BBa_K3725070) is composed of eight random base pairs, the biobrick prefix, the T7 promoter (BBa_J64997), the trigger sequence (BBa_K3725060), and the T7 terminator (BBa_K731721).

Figure 4. BBa_K3725010: Phytophthora Toehold w/ GFP Reporter

The Phytophthora Toehold w/ GFP Reporter (BBa_K3725010) is composed of four basic parts: the T7 promoter (BBa_J64997), the switch sequence (BBa_K3725050), a GFP reporter (BBa_E0040), and the T7 terminator (BBa_K731721).

Figure 5. BBa_K3725040: T7 Phytophthora Trigger Construct

The T7 Phytophthora Trigger (BBa_K3725040) is composed of four basic parts: the T7 promoter (BBa_J64997), the trigger sequence (BBa_K3725030), and the T7 terminator (BBa_K731721).

Figure 6. BBa_K3725022: Improved Fusarium Toehold w/ GFP Reporter

The Redesigned Fusarium Toehold w/ GFP Reporter (BBa_K3725022) is composed of four basic parts: the T7 promoter (BBa_J23100), the switch sequence (BBa_K3725080), a GFP reporter (BBa_E0040), and the T7 terminator (BBa_K731721).