Part:BBa_K2974101

Medium Promoter (BBa_J23106) Toehold GFP

BBa_J23106 Toehold eGFP is a construct designed to be utilized as a riboregulatory biosensor in conjunction with part BBa_K2550001. The part was assembled using a De-Novo Toehold Switch construct originally designed in the Collins Lab at the Massachuchets Institute of Technology and a medium-strength promoter BBa_J23106 of the Anderson Promoter family. Promoter BBa_J23106 is from the same parts collection as BBa_J23100 and was utilized to reduce overexpression due to diminished strength. Composite part BBa_K2974500 provides both a proof of concept for part BBa_K2974310 and characterization for promoter expression within the T7 promoter Toehold Ribosome Switch with the eGFP reporter expression system.

Description
Toehold switches are synthetic RNA strands that mimic messenger RNA sequences. They contain a complementary recognition sequence for a specific sequence of RNA stimuli , and a ribosomal binding site where a ribosome binds to initiate the translation of a reporter protein. The switch has a hairpin loop structure that is formed through binding to complementary sections of its own sequence. When in the presence of the complementary trigger sequence the hairpin loop opens to allow downstream expression. The Ribosomal Binding Site and starting sequence are concealed in the toehold switch until initiated. Switches can provide rapid, convenient, in-field detection that can be developed in both cellular systems and cell-free tests.

Results
The transformation of the team’s composite part BBa_K2974101, the BBa_J23106 (medium-strength) promoter with the Toehold eGFP construct, with pSB3C5, did not produce green fluorescent colonies. The absence of fluorescence was ideal, as the reporter protein eGFP should only be expressed in the presence of the trigger sequence. The red fluorescent colonies seen in both images is a result of the backbone, pSB3C5, which was originally cloned with Red Fluorescent Protein (RFP). Lambert iGEM obtained this stock of pSB3C5 from the iGEM Distribution Kit and proceeded to digest the plasmid with High Fidelity EcoRI and PstI restriction enzymes the red fluorescence observed on each plate indicates that the plasmid re-ligated to the RFP insert originally present in the pSB3C5 stock instead of the toehold construct during ligation with T4 ligase. The team inoculated colonies that were not producing RFP which appeared translucent after a 24 hour incubation period. Sequencing then confirmed that colony 3 (Figure 3) included the correct toehold sequence.

Figure 1: (Left) Transformation of the part BBa_K2974300, BBa_J23106 promoter Toehold eGFP construct, not under ultraviolet (UV) light. (Right) The same transformation of part BBa_K2974300 under UV light.

Figure 2. 1% gel with the 2-log ladder in well 5 and colony 3 in well 6. Both the insert (toehold - approximately 900 base pairs) and the vector (pSB3C5 - approximately 3000 base pairs) are present in colony 3. When this colony was sent out for sequencing, it aligned with the desired toehold sequence (Figure 3).

Figure 3. Sequencing results from IDT confirms that the toehold construct assembled with pSB3C5 aligns with the desired toehold sequence.

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 728