Results

mascot-results

JUMP Cloning of the inserts into pJUMP29A1:

The Golden Gate assembly (JUMP cloning) was successfully performed in the lab.

Figure 1 - JUMP cloning
Figure 1: Gel electrophoresis results of the cloned inserts. Wells 1 and 14 contained the 100bp and 1kb DNA ladder respectively. Wells 2-5 contained the miRNA while wells 6-9 and 10-13 contained toehold switches with the luciferase and mRFP reporter gene respectively. A mislabeling error occurred and that is why one of the luciferase and mRFP toeholds have switched places. We can also see that some of the pJUMP29A1 plasmid was not cut correctly, leaving the GFP intact, which can be seen in well 2 and 5 where 2 bands at approximately 700bp are observed.

Transformation of E.Coli:

The XL1-Blu E.Coli were successfully transformed with the pJUMP29A1 plasmid for propagation. XL1-Blu E.Coli were also transformed with mRFP and LacZ operon from the iGEM 2025 distribution kit.

The LacZ transformation failed, cells were dying too soon but the mRFP transformation was successful

Transformation of the products of the golden gate assembly was also successful, however, no cell growth was observed on the plate with mIRNA 363, therefore, it is not included in this section.

Figure 2 - XL1 BLu E.Coli transformed with miRNA 7850 containing plasmid
Figure 2: XL1 BLu E.Coli transformed with miRNA 7850 containing plasmid. Green colonies indicate unsuccessful transformations.
Figure 3 - XL1 BLu E.Coli transformed with miRNA 1306 containing plasmid
Figure 3: XL1 BLu E.Coli transformed with miRNA 1306 containing plasmid. Green colonies indicate unsuccessful transformations.
Figure 4 - XL1 BLu E.Coli transformed with miRNA 6529 containing plasmid.
Figure 4: XL1 BLu E.Coli transformed with miRNA 6529 containing plasmid. Green colonies indicate unsuccessful transformations.
Figure 5 - XL1 BLu E.Coli transformed with toehold 7850 + luciferase plasmid
Figure 5: XL1 BLu E.Coli transformed with toehold 7850 + luciferase plasmid. Green colonies indicate unsuccessful transformations.
Figure 6 - XL1 BLu E.Coli transformed with toehold 1306 + luciferase plasmid
Figure 6: XL1 BLu E.Coli transformed with toehold 1306 + luciferase plasmid. Green colonies indicate unsuccessful transformations.
Figure 7 - XL1 BLu E.Coli transformed with toehold 363 + luciferase plasmid.
Figure 7: XL1 BLu E.Coli transformed with toehold 363 + luciferase plasmid. Green colonies indicate unsuccessful transformations.
Figure 8 - XL1 BLu E.Coli transformed with toehold 6529 + luciferase plasmid
Figure 8: XL1 BLu E.Coli transformed with toehold 6529 + luciferase plasmid. Green colonies indicate unsuccessful transformations.
Figure 9 - XL1 BLu E.Coli transformed with toehold 7850 + mRFP plasmid
Figure 9: XL1 BLu E.Coli transformed with toehold 7850 + mRFP plasmid. Green colonies indicate unsuccessful transformations.
Figure 10 - XL1 BLu E.Coli transformed with toehold 1306 + mRFP plasmid
Figure 10: XL1 BLu E.Coli transformed with toehold 1306 + mRFP plasmid. Green colonies indicate unsuccessful transformations.
Figure 11 - XL1 BLu E.Coli transformed with toehold 363 + mRFP plasmid
Figure 11:XL1 BLu E.Coli transformed with toehold 363 + mRFP plasmid. Green colonies indicate unsuccessful transformations
Figure 12 - XL1 BLu E.Coli transformed with toehold 6529 + mRFP plasmid.
Figure 12: XL1 BLu E.Coli transformed with toehold 6529 + mRFP plasmid. Green colonies indicate unsuccessful transformations




Then after cloning with golden gate assembly, we have transferred the new constructed parts into BL21-de3 for protein expression, results can be seen in fig N


Figure 13 - BL21de3 E.Coli transformed with the 7850 toehold + mRFP
Figure 13:BL21de3 E.Coli transformed with the 7850 toehold + mRFP
Figure 14 - BL21de3 E.Coli transformed with 1306 toehold + mRFP
Figure 14: BL21de3 E.Coli transformed with 1306 toehold + mRFP
Figure 15 - BL21de3 transformed with the 363 toehold + mRFP.
Figure 15:BL21de3 transformed with the 363 toehold + mRFP.
Figure 16 - BL21de3 E.Coli transformed with the plasmid containing the 6529 toehold + mRFP
Figure 16: BL21de3 E.Coli transformed with the plasmid containing the 6529 toehold + mRFP

miRNA detection by toehold switches:

Our team has not observed mRFP expression when the toehold switch was in the cell free system with the miRNA present. This indicates that some part of the toehold does not function as it was expected. After careful analysis, our team hypothesises that it is the expression of the mRFP reporter gene, rather than the failure for the toehold to unfold when miRNA is present.

Figure 17 - The results after repeated mRFP spectrophotometry readings.
Figure 17: The results after repeated mRFP spectrophotometry readings. Wells A1-A6 contained reactants while other wells remained empty. Wells A1 and A2 contained negative and positive control respectively. Well A3 contained the miRNA 7850 and its complementary toehold, well A4 contained miRNA 1306 and its complementary toehold, well A5 contained dthe mIRNA 363 and no toehold switch and the last well - A6 contained the miRNA 6529 and its complimentary toehold.

Future Research:

In this study we found that the designed toehold did not produce a signal when the miRNA was present. In future studies that aim to mirror this experimental method, we suggest that it may be beneficial to add a longer linker sequence between the toehold region and the mRFP sequence. One may achieve reduction of unwanted binding in order to increase sequence specificity towards the miRNA via codon changing, enabling simultaneous preservation of the encoded amino acid sequence.

Despite the results of this study not yielding a concrete diagnostic technique for LTB, they hold value by enabling the identification of potential toehold modifications. The effectiveness of these adjustments are still to be assessed, and we believe that this may provide a progressive avenue for future research, and serve the troubleshooting of improved toehold system design.