Team:Thessaloniki/Contribution

☰







Contribution






Overview


During this year’s journey, our team made a considerable contribution to future iGEM teams, mainly through the introduction of different parts to the iGEM registry. These parts are toehold switch sequences and while they are focusing on Pancreatic Ductal Adenocarcinoma (PDAC), with the right modifications, they can be used for the diagnosis of other diseases too. In order to achieve PDAC early diagnosis, we chose to design toehold switches for the detection of three microRNAs; hsa-miR-143-3p, hsa-miR-30e-5p, and hsa-miR-1246. For each of these microRNAs, we designed multiple toehold switch sequences by improving the previously used Design Software in order to achieve better thermodynamic abilities. During the competition, a lot of iGEM teams, that used toehold switches, were facing difficulties with the leakage signal. In our toeholds, we managed to remove the pre-RBS sequence in the loop and in this way to reduce the leakage signal caused by weak bonds in the body of the hairpin. This means that we managed to minimize the fluorescence produced by the eGFP in the absence of the microRNAs satisfactorily. We hope that our toehold switches will contribute to the future iGEM teams as a groundwork for further improvement of toehold stability.

For more details visit the following parts (Table 1) in the iGEM registry.
BBa_K3727018 Composite T7_Promoter_Toehold_Switch-143-3p (3)_ eGFP_T7_Terminator
BBa_K3727019 Composite T7_Promoter_Toehold_Switch-143-3p (4)_ eGFP_T7_Terminator
BBa_K3727020 Composite T7_Promoter_Toehold_Switch-30e-5p (1)_ eGFP_T7_Terminator
BBa_K3727021 Composite T7_Promoter_Toehold_Switch-30-5p (2)_ eGFP_T7_Terminator
BBa_K3727022 Composite T7_Promoter_Toehold_Switch-30-5p (3)_ eGFP_T7_Terminator
BBa_K3727023 Composite T7_Promoter_Toehold_Switch-30-5p (4)_ eGFP_T7_Terminator
BBa_K3727024 Composite T7_Promoter_Toehold_Switch-30-5p (5)_ eGFP_T7_Terminator
BBa_K3727025 Composite T7_Promoter_Toehold_Switch-30-5p (6)_ eGFP_T7_Terminator
BBa_K3727030 Composite T7_Promoter_Toehold_Switch-1246 (3)_ eGFP_T7_Terminator
BBa_K3727031 Composite T7_Promoter_Toehold_Switch-1246 (4)_ eGFP_T7_Terminator



Methods


Our toeholds were designed using NUPACK and ViennaRNA software, following the basic rules of toehold switches' design. However, they are composed of three extra subsequences and they are missing the pre-RBS area. These modifications were made to make our toeholds more stable:
  • 1. In the Sensory Domain: codon "GGG" followed by unpaired and paired bases complementary to the miRNA sequence.
  • 2. In the Regulatory Domain: In the Regulatory Domain: codon "GUA" followed by the sequence "GUGUGU", the RBS sequence followed by "ACACAC" and the start codon "AUG"
These changes were made because of our toeholds' shorter size. Our toeholds are designed to detect microRNAs, which are small sequences of RNA, and thus the sensory domain of our toeholds is smaller. Because of the sensory sequence's smaller size, the existence of the pre-RBS area caused problems with the secondary structure and had to be removed. For each microRNA, we designed multiple toeholds switches, with different RBS and Linker sequences.

In order to test our sequences, we first resuspended the g-Blocks and then we followed a cloning method to add our sequences into the plasmid vector.

For more details please visit our “Experiments” page:

Experiments




After the transformation of competent Escherichia coli cells with the recombinant plasmid, we tested our toehold switches in the cell-free system (NEB - PURExpress® In Vitro Protein Synthesis Kit). We tested our sequences both with and without the presence of the trigger microRNA and we measured the fluorescence produced.



Results


Our contribution this year consists of the following parts:
Part Name Trigger RNA RBS Sequence Function
BBa_K3727018 miR-143-3p RBS Green Low leakage signal
BBa_K3727019 miR-143-3p RBS DHFR Low leakage signal
BBa_K3727020 miR-30e-5p RBS DHFR High leakage signal
BBa_K3727021 miR-30e-5p RBS Green High leakage signal
BBa_K3727022 miR-30e-5p RBS DHFR High leakage signal
BBa_K3727023 miR-30e-5p RBS Green Low leakage signal
BBa_K3727024 miR-30e-5p RBS DHFR Low leakage signal
BBa_K3727025 miR-30e-5p RBS Green Low leakage signal
BBa_K3727030 miR-1246 RBS Green Low leakage signal
BBa_K3727031 miR-1246 RBS DHFR Low leakage signal
Table 1: List of Toehold Switches parts we contributed to iGEM Registry characterized by the Trigger RNA they are complementary to and the RBS containing.
RBS Green is the RBS sequence that Green et al. introduced in 2014 [1], while RBS DHFR is the RBS sequence suggested by the manufacturer of the cell-free system we used [2]. Generally, the toeholds with the DHFR RBS showed better results, while the majority of our toeholds showed very little leakage signal. You can see the detailed fluorescence results of our toehold switches on our Toehold Switches Lab Book:

Wet Lab Notebook



References