This week was all about moving into our new lab and getting everything organized!
  • Elia, Lena, Lisa, Malek:
  • We started running PCRs to amplify linear DNA templates we needed for our experiments.
  • To see whether our lysates are able to express β-Galactosidase, we ran plate-reader experiments that took a bit longer than anticipated, due to the plate-reader's lack of cooperation.
  • We prepared our own competent cells and transformed them with a vector containing part of the β-Gal gene that can be found on the iGEM plates.
  • Worked on the fabrication of MITOMI chips that can be attached to epoxy-coated slides.
  • Felix, John, Matteo, Tim:
  • Worked on the fabrication of 50 epoxy-coated glass slides for our experiments.
  • Four of the epoxy-coated glass slides were spotted with Cy3/Cy5-labeled aptamer dilutions.
  • An experiment was carried out using MITOMI chips to determine whether or not the thrombin aptamers 1 and 2 were able to bind biotinylated-thrombin. As expected, we observed a fluorescent signal from Cy3/Cy5 labeled anti-thrombin aptamers bound to immobilized biotinylated thrombin in the button areas. This experiment was repeated three times and a calibration curve for both aptamers was performed on the same chip.
  • Lab notebooks:
  • 170703_Dh5a M15 lysates
  • 170712_LacZa_transformation
  • 170713_M15-T7 and Top10-gamS lysates
  • 170713_Competent_cells
  • 170713_Glass_slide_preparation
  • 170713_Glass_slide_spotting
  • 170714_Aptamer_Binding
  • 170716_E.coli-T7 GFP DNA
17/07/2017 - 23/07/2017
This week was all about planning further experiments...
  • Felix, John, Matteo, Tim:
  • We started by the fabrication of new MITOMI chips.
  • We planned a competition MITOMI experiment to check whether there is competition in the binding of the thrombin aptamers 1 and 2. Sadly, it didn't work the first time, in part because the concentrations were too low, consequently, saturation could not be reached.
  • Elia, Lena, Lisa, Malek:
  • Non-biotinylated alpha human thrombin was ordered.
  • We brainstormed on the fabrication of a functional lateral flow immuno-assay using two anti-thrombin aptamers.
  • Lab notebooks:
  • 170718_PCR lacZa_toehold
  • 170718_Platereader_GFP
  • 170719_Calibrate_platereader
  • 170719_GFP_platereader
24/07/2017 - 30/07/2017
Wrestling with PDMS chip making and baby's first steps... Representing iGEM EPFL in Cambridge!
  • John, Matteo, Tim:
  • We continued with MITOMI chip making and failed... miserably. But then a ray of hope, our first chip that did not delaminate. And so it began!
  • We learned how to use the data analysis program and determined the best statistical model for a saturation curve.
  • Plotted our first saturation curve for both the thrombin aptamers 1 and 2.
  • Lisa, Lena, Felix:
  • We went to Cambridge to attend the Open Plant Forum, take part in a workshop on cell-free in high schools and made first contact with iGEM Oxford
  • Lab notebooks:
  • 170728_Pure_express_test
31/07/2017 - 6/08/2017
MITOMI chip sandwich assay and titration of ssDNA in lysate
  • Felix, John, Matteo, Tim:
  • Sandwich assays with Cy3 and Cy5 labeled aptamers were performed in order to determine whether or not both could simultaneously bind thrombin.
  • Elia, Lena, Lisa, Malek:
  • We compared if there was a difference between adding pure GamS, that we ordered, and lysate containing GamS produced by One Shot TOP10 Chemically Competent E.coli cells.
  • Short and long ssDNA trigger sequences were incubated with LacZ coding toehold in PURE and T7M15 (TOP10GamsS) lysate. Results were... well... inconclusive, but we have nice pictures!
  • Lab notebooks:
  • 170731-Top10GamS-BL21-GFP
  • 170805_Efficiency-BL21-Top10-addedGamS
  • 170801_M15_Characterization
  • 170801_Sandwich_Assay
  • 170806-Ratio_Top10_gamS
7/08/2017 - 13/08/2017
Chip sandwiches, a new toehold and titration a gogo...
14/08/2017 - 20/08/2017
Pulldown assays and freeze-drying our lysate
21/08/2017 - 27/08/2017
Magnets, beads, streptavidin, LacZ toeholds and titration, titration, titration...
28/08/2017 - 03/09/2017
And more titration! And a few pulldowns...
04/09/2017 - 10/09/2017
Pulling down, improving lysate reactions, new aptamer design and testing in serum
  • Tim:
  • Following negative results, pulldown experiments were performed with different biotinylated compounds to check if our streptavidin beads were still functional. However, the substrates used were in quantities too low to determine anything.
  • However, we finally did a titration of Cy5 labeled aptamer using the magnetic beads and determined the detection limit for 60-18[29]a aptamer.
  • Elia, Lena, Lisa, Malek:
  • We tested out whether or not adding Buffer Z to lysate/toehold/trigger solutions improved reaction kinetics and output, results were good and this proved that the extra buffer was unnecessary.
  • John, Matteo:
  • We both designed a new trigger-aptamer (aka thrombin aptamer 2 + trigger extension) with the help of NUPACK for its secondary structure.
  • We tested out whether or not the new design correctly bound thrombin and correctly triggered the toehold. And the experiment was performed again using serum as a medium.
  • Lab notebooks:
  • 170904_Test_lysates
  • 20170905-06_beads
  • 20170907_beads
  • 170909_bufferZ_titration
  • 170909_Sandwich_assay_serum
11/09/2017 - 17/09/2017
New Trigger-toehold failure, double-bind success! Designing yet another aptamer... And attending a summer school
  • Elia, Lena:
  • We tried to ligate and amplify the new 32B toehold and its trigger. Only the first overlap PCR worked.
  • Malek, Lisa:
  • We represented iGEM EPFL at the Summer School Shaping the Future of Bioengineering, including making a poster, having a presentation about iGEM and aptasense, and taking part in a poster session.
  • John, Matteo:
  • The design of a new anti-PDGF aptamer with 27B trigger was the focus of the week. Upon receiving it, we began testing, on MITOMI chip, whether or not it fulfilled its expected functions.
  • Tim:
  • Biotinylated thrombin, aptamer double bind assays were performed this week.
  • Lab notebooks:
  • 20170911_beads
  • 20170914_beads
  • 170908_32B_new_toehold
18/09/2017 - 24/09/2017
Correcting titration mishaps, testing the time factor in adding trigger, PDGF aptamer testing and pulldown mess up
  • Elia, Lena, Lisa, Malek:
  • Titration of aptamer, 10 to 0.5 uM, in PURE and M15-T7 lysate was performed and repeated, giving good results. We finally managed to titrate short and long ssDNA in PURE and M15-T7.
  • Moreover, we tested out whether or not adding trigger at different time intervals after the beginning of reactions had an impact on the output quality and intensity. The results obtained were good.
  • We cloned the first part we wanted to submit, GamS, into the iGEM backbone.
  • John, Matteo:
  • The team continued to test out whether the anti-PDGF aptamer correctly bound its target protein and whether or not the toeholds were correctly triggered. Of course all testing was performed on chip and the problems encountered prevented us from obtaining good results.
  • Lab notebooks:
  • 170918_ssDNAshort_titration
  • 170918_aptamer_and_ssDNAlong_titration
  • 170922_ssDNAshort_titration
25/09/2017 - 01/10/2017
Beads: Thrombin titration, magnetic bead sandwich assay. Freeze-dry lysate resuspension, ordering new Hepatitis C toeholds and triggers.
  • Tim:
  • Magnetic beads were used to perform two very different tests this week: a titration of thrombin and a sandwich assay with a trigger extended aptamer. The thrombin titration was successful and wielded great results, unlike the sandwich assay.
  • Natalija:
  • After finding unique sequences for Hepatitis B, C and G, the software was used to generate 4 toehold-trigger pairs for Hepatitis C.
  • Elia, Lena, Lisa, Malek:
  • We resuspended our lysate after freeze-drying and the results were inconclusive.
  • We also tested out the expression of the LacZ-α toehold in various different lysates.
  • The team ordered 32B toehold and corresponding Hepatitis C triggers to prove that our system is modular.
  • Moreover, we successfully performed a Gibson assembly of GamS into the iGEM backbone.
  • John, Matteo:
  • We finally managed to prove that our aptamer pair binds PDGF in a sandwich assay and we tested out whether or not the aptamer extended with trigger could correctly activate the toehold.
  • Lab notebooks:
  • 20170927_beads
  • 20171001_beads
02/10/2017 - 08/10/2017
Sequencing and improving part characterization, testing the entire project on chip and wiki
  • Elia, Lena, Lisa, Malek:
  • We sequenced the GamS that was inserted into the iGEM backbone to check if all was correctly done.
  • We decided to improve the characterization of the LacZ toehold in cell free medium, thus we tried out the system in various lysates.
  • John, Matteo:
  • We moved on to testing the entire project on chip. Expressing toehold on the chip using a substrate of β-Galactosidase. The whole system was tested in Top10-GamS and M15-T7 lysate. Unfortunately there was some leakage on the chip, ruining the experiment.
  • Tim:
  • Started discussing how pulldown sandwich assays should then include the flowing of lysate with toehold, to see if said toeholds could correctly be triggered by sequences present on positive pulldowns.
  • This week the entire team's focus gradually started shifting towards the elaboration of the wiki.
09/10/2017 - 15/10/2017
Gibson and transformation, amplification, testing software toeholds, repeating chip experiment and failing and wiki, wiki
  • Elia, Lena:
  • We did Gibson assemblies of GamS followed by the transformation of cells with the assembled plasmids. Failure was on the menu.
  • Lisa:
  • The amplification of the aptamer extended with a T7 promoter and complementary trigger sequence was the main focus of the week. However, said amplification was unsuccessful.
  • Malek:
  • The various toeholds generated by the software needed to be tested to confirm that it functioned correctly, and indeed it does. This week also included giving a hand with colony PCRs and PCRs for the Gibson assembly.
  • John, Matteo:
  • We repeated last week's experiment with a slight difference, a stronger buffer. This time we had signal everywhere, even where the trigger extended aptamer was absent.
  • With the wiki-freeze approaching the wiki comes more and more into focus.
  • Lab notebooks:
  • 171012_t7_aptamer_trigger
16/10/2017 - 22/10/2017
Chips: same, same but different, Gibson a gogo, toeholds and triggers, and wiki, wiki, wiki
  • John, Matteo:
  • We redid the experiment from the previous weeks, one caveat, we reduced the FDG concentration by a factor of 2, to decrease signal intensity. Since there was still signal in the control without the trigger, so we decided to use PURE instead of our own lysate.
  • Elia, Lena:
  • We spent the week doing various Gibson assemblies and subsequently transforming cells with the resulting plasmids (iGEM backbone with LacZ-α, 27B or toehold B) to ensure success. Often that was not the case.
  • Lisa:
  • Continuing attempts at amplifying the aptamer extended with T7 and complementary trigger that were ultimately unsuccessful was the main focus of the week.
  • Malek:
  • This week, the aim was to help out with amplification as well as colony PCRs.
  • Tim:
  • Testing out the hypothesis for the pulldown assay with trigger extended aptamer followed by the addition of lysate with toehold proved to be inconclusive to the naked eye. However, a nanodrop testing of the final solutions showed extreme expression quantities in the assay containing trigger. The team scratched their heads as to what they should conclude from such results.
  • Finally, the entire delved even deeper into the depths of wiki elaboration, now the main priority.
  • Lab notebooks:
  • 20171022_beads
23/10/2017 - 29/10/2017
Sandwich pulldown fury, testing for trigger-toehold instability and wiki to infinity and beyond
  • Elia, Tim:
  • We redid the pulldown experiment with trigger extended aptamer followed by the addition of lysate containing toehold to no avail. Since our controls behaved peculiarly we decided to simply test the trigger-toehold system without beads and obtained good results.
  • Lena:
  • Successfully inserting the remainder of our parts into the iGEM backbone was the highlight of the week.
  • And the wiki...
  • Lab notebooks:
  • 20171023_beads
  • 20171025_beads
  • 20171026_beads
  • 20171027_beads
30/10/2017 - 1/11/2017
The storm before the calm...
  • Everyone:
  • Wrapping up the final few experiments that needed to be done and losing sleep to an unhealthy degree in order to complete this wiki and prove that the project works. But it was worth it.
  • We're proud of our work and would do it all over again.
  • Thanks team!
  • Lab notebooks:
  • 20171030_beads
  • 20171101_beads

  • The end.