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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.
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The transformation of the team’s composite part BBa_K2974700, BBa_J23100 (strong) promoter and Toehold eGFP construct, in pSB3C5, produced both translucent and red pigmented and fluorescing colonies. The red fluorescent colonies seen in the image is a result of the backbone, pSB3C5, which was obtained from the iGEM Distribution Kit and was originally cloned with RFP, re-ligating with its original insert rather than the toehold. The team inoculated the colonies that were not producing RFP. Sequencing then confirmed that colony 3 included the correct toehold sequence.  
 
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Revision as of 17:46, 21 October 2019


Strong Promoter (BBa_J23100) Toehold GFP

BBa_J23100 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 strong T7 promoter BBa_J23100 from the Styczynski Lab at the Georgia Institute of Technology. The Standard Toehold Switch was assembled with eGFP (BBa_E0040) as a reporter protein. The T7 promoter Toehold Ribosome Switch with LacZ expression (BBa_K2550000) constructed by the 2018 iGEM team utilized strong promoter BBa_J23100 and experienced significant overexpression as a result. Composite part BBa_K2974700 provides 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_K2974700, BBa_J23100 (strong) promoter and Toehold eGFP construct, in pSB3C5, produced both translucent and red pigmented and fluorescing colonies. The red fluorescent colonies seen in the image is a result of the backbone, pSB3C5, which was obtained from the iGEM Distribution Kit and was originally cloned with RFP, re-ligating with its original insert rather than the toehold. The team inoculated the colonies that were not producing RFP. Sequencing then confirmed that colony 3 included the correct toehold sequence.


Figure 1. Transformation of the part BBa_K2974700, the BBa_J23100 promoter Toehold eGFP construct, with pSB3C5 under UV light.


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