January - March
Students were invited to participate in weekly iGEM brainstorming sessions. Over the course of two months we narrowed down topics and ended up with a team of eleven students and the beginning of Colourectal.
March - April
From March onwards the team entered the Team-based Synthetic Biology Project Design course. Over the course of eight weeks we investigated what makes a great iGEM project, and spent time researching and developing the Colourectal project and its subprojects. This cumulated in each of us presenting our project proposals at the end of April.
May - October
Week 1
Having developed a proper research plan we set out to perform our research in the lab. Learn more about this in each of the personal notebooks! Besides lab work we also started working on our education material for the upcoming weeks.
Week 3
We gave a workshop about bacteria to primary school kids. This formed the basis for our lovely merch and the start of our education.
Week 5
Team building is important, so we took off to Groningen for a weekend of fun activities!
Week 7
Now human practices really started to kick into overdrive. We gave two school workshops, and spoke with the RIVM, Erasmus Medical Centre, and Icos about our project.
Week 8
We made a vlog together with Student Challenges about our project. We also interviewed Danielle Grashuis, Dr. Markus Gwiggner, and Stichting Darmkanker for our human practices. Additionally, we went to the European iGEM Meetup in Hamburg over the weekend where we connected with lots of teams.
Week 9
This week we interviewed Ray Brouwer, and Lizette de Vries from Stichting Darmkanker. Moreover, we organized the Dutch iGEM meetup together with the TU Eindhoven team and the Dutch centre of living technology.
Week 10
We interviewed a colorectal cancer patient, Scope, the RIVM, and Leiden Medical Centre for our project.
Week 11
After connecting with them at the Dutch meetup we kicked off our partnership with the Groningen iGEM team!
Week 13
We interviewed NIZO and Johnson & Johnson for advice on our project. We make the first contact with KU Leuven for a new collaboration.
Week 14
This week we spoke with OnePlanet about colorectal cancer diagnostics.
Week 15
We interviewed Beatrijs Haverkamp, a philosophy researcher.
Week 16
We started working on our iGEM project promotion video, and checked in on our partnership progress with Groningen. Additionally, since we can never get enough input for our project, we spoke with MEB, the RIVM, and COGEM.
Week 17
We submitted our iGEM project promotion video, and interviewed Inscripta about biosecurity.
Week 18
We held our stakeholder meeting to share what we learned from them, and gain more shared insights to incorporate further.
Week 20
We launched our cookbook in the public library of Wageningen. Delicious! The focus of our working days slowly starts to shift from lab work to wiki writing.
Week 21
We had another interview with NIZO about the implementation of our living diagnostic.
Week 22
Following up on the interview the week before, we spoke with another expert from NIZO about other potential probiotics that could be adapted for Colourectal in the future.
Week 23
The wiki deadline pushes us to work hard and make sure all pages are in top form.
Week 1
Design constructs and order parts.
Week 2
Perform in silico modelling of the chromoproteins in Alphafold.
Week 3
Start assembly of chromoprotein constructs.
Week 4
Assembly of chromoprotein constructs.
Week 5
Troubleshooting chromoprotein expression.
Week 6
Cloning to switch to a new chromoprotein backbone plasmid.
Week 7
Further assembly of chromoprotein constructs.
Week 8
Assembly of chromoprotein constructs.
Week 9
Troubleshooting chromoprotein expression (again).
Week 10
Assembly of the MMP-9 expression plasmid.
Week 11
Switch to the E. coli BL21 strain to improve chromoprotein expression.
Weeks 12 to 15
Assembly of flip-chromoproteins.
Week 16 & 17
Holiday break!
Week 18
Assembly of flip-chromoproteins.
Week 19
Troubleshooting the flip-chromoproteins. Also, lots of wiki writing from here on.
Week 20
Troubleshooting the flip-chromoproteins.
Week 21
Western Blot experiment to verify MMP-9 expression.
Week 22
Fluorescence experiment of flip-chromoproteins.
Week 1
Literature study and ordering the gas vesicle plasmid from AddGene.
Week 2
Prepared media and competent DH10B cells in addition to more literature study.
Week 3
Amplified and purified the acoustic reporter gene (ARG) plasmid and backbone for the inducible promoter systems.
Week 4
Amplified the parts for the inducible promoter systems.
Week 5
Started assembly of inducer system plasmid backbone, ordered gBlocks with promoter systems.
Week 6
Made competent Escherichia coli Nissle 1917 (EcN). Transformed ARG plasmid into EcN.
Week 8
Made more LB, assembly of inducable promoter system plamids.
Week 9
Performed colony PCR on the backbone.
Week 10
Induced gas vesicle production with IPTG in EcN containing ARG plasmid, tried to confirm gas vesicle production with float test.
Week 11
Changed design of inducible promoter system plamid backbone after input from other supervisors.
Week 12
Assembly of new inducible promoter system plasmid backbone.
Week 13
Assembly of new inducible promoter system plasmid backbone. Amplified bacterioferritin (bfr) gene from plasmid found in iGEM registry, and ARG backbone.
Week 14
Colony PCR on inducible promoter system plasmids and sent for sequencing. Introduced bfr into ARG backbone, transformed into DH10B. Tried induction and float test again for EcN with ARG plasmid.
Week 15
Assembly of inducible promoter system plasmids. Redesigned Bfr plasmid, amplified bfr again.
Week 16
Colony PCR on inducible promoter system plasmids and sent for sequencing. Transformed new bfr plasmids into DH10B. Induced gas vesicle production using IPTG in EcN with ARG plasmid again, left cultures on bench instead of doing the float test.
Week 17
Plate reader experiment with one of the inducable promoter systems in EcN. Decided to stop the bfr and ARG experiments due to time constraints.
Week 18
Plate reader experiment with different inducible promoter system.
Week 19
Ordered gBlocks of more inducible promoter systems.
Week 20
Data analysis of plate reader experiments. Also started on lots of writing and preparation for the wiki.
Week 21
Assembly of new inducible promoter system plasmids.
Week 22
Something went wrong with the assembly, not enough time to redesign them unfortunately.
Week 19
Ordered gBlocks of more inducible promoter systems.
Week 1
Working out how to structure my code.
Week 2
Literature research for the model.
Week 3
Recreating the model of Rafael Muñoz-Tamayo from the paper and learning how to work with it.
Weeks 4
Fit the model to data from papers.
Week 5
Refining the optimization strategy.
Week 6
Wrote the script to report results.
Week 7
Organizing myself and the data.
Week 8
Presented progress in the Computational Systems and Synthetic Biology meeting.
Week 9
Experimented with some multidimensional data visualisation.
Week 10
Messed with interpolating a dose-response curve.
Week 11
Further literature research.
Week 12
Worked on a sensitivity analysis script.
Week 13
Scripting analysis results visualisation.
Week 14
Processed data from plate reader experiments.
Week 15
Attempted to fit the model to data obtained in the lab by Bas.
Week 16
Restructuring code and data.
Week 17
Fit growth data separately in order to simplify optimisation.
Week 18
Finally found a good fit to the data!
Week 19
Combination with the asRNa model, and performed sensitivity analysis.
Week 20
Organising myself and writing for the wiki
Week 21
Wiki development, writing a script for standardised graphs, and writing up the modelling sections.
Week 22
Wiki development, gathering data, and processing graphs.
Weeks 1 & 2
Designing in silico.
Week 3
Amplification and purification of parts for the chimeric mucin sensor.
Week 4
Golden gate assembly of the chimera and homology arms for the EnvZ KO of E. coli Nissle.
Week 5
Plate reader experiment to test temperature sensitive promoters.
Week 6
Troubleshooting EnvZ KO in E. coli Nissle.
Week 7
Golden gate assembly of the chimeric receptor with a strong RBS and a strong promoter.
Week 8
Troubleshooting of the previous weeks chimeric receptor assembly.
Week 9
Golden gate assembly of the chimeric receptor in a second cycle of cloning.
Week 10
Amplification and cloning of OmpC with GFP
Week 11
Designing an experiment to test the chimeric receptor and in silico design of ccdA-B temperature sensitive kill-switch.
Week 12
Amplification of ccdA, ccdB and the temperature sensitive cspA promoter.
Week 13
Testing the chimeric receptor in E. coli Nissle with mucin in a plate reader experiment.
Week 14 & 15
Some well-deserved time off!
Week 16
Amplification of ccdA, ccdB and the temperature sensitive TlpA promoter.
Week 17
Data analysis of the plate reader experiment testing the chimeric receptor and different mucin concentrations.
Week 18
Testing the two temperature sensitive constructs with cell death assays.
Week 19
Troubleshooting the cell death assay results.
Week 20
Cloning and testing of the pTlpA construct in the backbone we received from the Groningen iGEM team as part of our partnership.
Week 21
Data analysis and discussion of results.
Week 22
Lots of writing for the team wiki.
Weeks 1 & 2
Designing in silico and ordering primers.
Week 3
Amplifying the pSEVA258 backbone.
Week 4
Integrate restriction sites for integration of plasmid.
Week 5
Digestion of restriction sites.
Week 8
Troubleshooting the integration plasmid.
Week 9
Redesigning and ordering new primers.
Week 10
Extension PCR of the fusion proteins.
Week 11
Cultivation of k-12, MG1655 and extension PCR fusion proteins.
Week 12
Prepare binding assay test and extension PCR fusion proteins.
Weeks 13 to 15
Extension PCR of fusion proteins and binding assay test.
Weeks 16 & 17
Gone for a nice holiday.
Week 18
Ligation of the pSEVA258 backbone and inserts. Made competent cells and transformed them.
Week 19
Sequence colony PCR plasmids.
Weeks 20 & 21
Troubleshoot colony PCR, use other primers. Send plasmids for sequencing. Also there’s lots of writing to do for the wiki.
Week 22
Try a biding assay of empty EcN, empty vector and both wildtypes on CEACAM6, integrin α5β1 and Caco-2 cells.
Weeks 1 & 2
Designing experiments.
Week 3
Designing more experiments and further primers.
Week 4
I received the Caco-2 cell lines.
Week 6
Freezing Caco-2 cells for storage.
Week 8
Test experiment for the Caco-2 cell collection.
Week 9
Test experiment for Caco-2 cell RNA isolation. Determination of conversion of OD600 to CFU/mL for E. coli Nissle.
Week 10
Preparation of heat inactivated bacteria.
Week 11
Sample collection for RNAseq of the Caco-2 samples.
Week 12
RNA isolation of Caco-2 cells.
Week 17
Caco-2 RNA was send for sequencing.
Week 18
Sample collection for RNAseq of bacterial samples.
Week 19
Cytotoxicity experiment on Caco-2 cells.
Week 20
RNA isolation of bacterial cells.
Week 21
RNA isolation of bacterial cells. Also, lots of writing for the wiki.
Week 22
Assessment of bacterial RNA, samples of which were send for sequencing. Analysis of the RNAseq of Caco-2 cells. Western Blot analysis of MMP-9 presence. Cytotoxicity assay of chromoproteins on Caco-2 cells.
Weeks 1 & 2
Designing experiments in silico.
Week 3
Design and order primers.
Week 4
Assembly of the first level of plasmid.
Week 5
Assembly of the full expression system with pLac.
Week 6
Working to fix the leakiness of the pLac promoter.
Week 7
After working on the plasmid there was no expression of the chromoprotein anymore, so I switched to a constitutive promoter.
Weeks 8 to 11
Cloning the pTarget for the E. coli Nissle rhamnose KO over two attempts.
Week 12
Switch to a constitutive promoter and GFP for the secretion assay.
Week 14
Primer design and rounding off the double rhamnose KO.
Week 15
Assembly and cloning of the constitutive promoter with GFP.
Week 16
Secretion assay of GFP.
Week 17
Cloning the rhamnose inducible plasmid.
Week 18
Rhamnose induced secretion assay.
Week 21
Chromoprotein purification.
Week 22
Testing to see if pink chromoprotein is visible in fake poop.
Weeks 1 to 10
Recreating the model of Rafael Muñoz-Tamayo from the paper and learning how to work with it.
Weeks 11 to 14
Implementing E. coli Nissle in the model.
Week 15
Perform adherence simulations.
Week 16
Adjusting the E. coli Nissle model.
Weeks 17
Perform new adherence simulations with the improved model.
Weeks 18
Adjust the uptake rate in the model.
Weeks 19
Perform dosing strategy simulations.
Week 20
Perform kill-switch simulations.
Weeks 21
Perform the final simulations and wrap up wiki writing.
Weeks 22
Work hard on the wiki development.
Weeks 1 to 4
Designing constructs in silico.
Weeks 5 & 6
Construction of the lactate sensing construct and a KO plasmid.
Week 7
Troubleshooting primers.
Weeks 8 & 9
Golden gate assembly of the chimera and homology arms for the EnvZ KO of E. coli Nissle.
Weeks 10 & 11
Vacation time!
Weeks 12 & 13
Creating the E. coli Nissle lldD KO.
Weeks 14 & 15
Plate reader experiments with the lactate sensor.
Week 16
Working hard on the iGEM project promotion video.
Week 17
Making an E. coli Nissle ykgF KO.
Weeks 18 & 19
Running plate reader experiments. Lots of writing for the wiki too!
Weeks 20 & 21
Making an E. coli Nissle nldH KO.
Week 22
Final plate reader experiments.
Week 1
In silico design of constructs.
Week 2
Order primers for cloning of inducible systems, GoldenGate assembly and DH5a transformation.
Week 3
Plasmid sequence verification.
Weeks 4 & 5
Testing of inducible systems in EcN.
Week 6
Plate reader growth experiment.
Week 7
Troubleshooting inducible system plasmids and change of strategy aspirin -> rhamnose.
Week 8
Cloning of CRISPR-Cas plasmid and ordering spacer oligos.
Week 9
Testing the rhamnose inducible system.
Week 10
Plate reader growth experiment .
Week 11
Cloning of the spacer in CRISPR plasmid.
Week 12
Cloning of the spacer in CRISPR plasmid and plate reader growth experiment.
Week 13
Spacer cloning was successful so I started testing the rhamnose-inducible CRISPR-Cas system in EcN.
Week 14
Plate reader growth experiment CRISPR-Cas in EcN.
Week 15
A well-deserved holiday break.
Weeks 16 & 17
Testing the CRISPR-Cas system.
Weeks 18 & 19
Running plate reader experiments for the kill-switch.
Weeks 20 & 21
Analysing data from the plate readers, and writing for the wiki.
Week 22
Lots more writing for the wiki!
Week 1
Preparing LB, plates, and competent cells.
Weeks 2 & 3
Designing in silico.
Week 4
Gathering parts from the iGEM registry.
Week 5
Amplification and purification to prepare the parts for assembly.
Week 6
Golden gate cloning for the double repression plasmid and transformation.
Week 7
Troubleshooting the previous weeks experiments. In the end two positive colonies were obtained.
Week 8
Sequencing the double repression plasmid. Golden gate cloning of the three step plasmid.
Weeks 9 to 12
Troubleshooting of the three step system plasmid.
Week 13
Introduction of sgRNA into the pTarget plasmid.
Week 14
Successful introduction of sgRNA in pTarget. Identified an important problem in the three step system plasmid.
Week 15
Construction of alternative three step plasmids with three separate fluorescent proteins and one complete construct with a low copy number.
Week 16
Screening of constructed plasmid and selection of colonies.
Week 17
Introduction of specific spacers for the three step system.
Week 18
Introducing double repression plasmid into E. coli Nissle 1917 KO. Plate reader experiment with luciferase and spacer introduction.
Week 19
Plate reader experiment to test the reporters and introducing spacers. Make a start at writing for the wiki.
Week 20
Introducing three gene plasmids into E. coli Nissle 1917 strain with double repression plasmid in it. Platereader experiments luminescence..
Introducing three gene plasmids into E. coli Nissle 1917 strain with double repression plasmid in it. Platereader experiments luminescence..
Week 21
Plate reader experiments luminescence, data analysis and fluoresence RFP.
Week 22
Plate reader experiments of the three step system with RFP fluorescence.