Notebooks

Wetlab Notebook

When working in the laboratory it is vital to document all the experiments, specially when working in a group, since you might have to continue someone else task. At the beginning of our laboratory work, we recorded each task, the team member who completed it, the date, results, and observations. Later on, we decided to work with mini-projects* to have a more decentralized and structured way of working. For better tracking, we began organizing information in the notebook according to each mini-project number. On this page, you can find the notebook as well as a description of each mini-project. However, if you want more information about the experiments, check out our experiments page .

*To have more information about our mini-project methodology visit our contributions page contributions page .

MP#1: Saving Private Ryan
  • Goal: Most of the short peptide sequences that were introduced by inverse PCR were not positive after transformation. This project aimed to fix those in less than 10 days (about 1 and a half weeks) and detect colonies expressing these short peptides.
  • Dates: 15/07/2024 -> 24/07/2024
  • Results: The primers used had mutations so we developed a new strategy based on two overlapping primers with repeated sequences in both ends so that they circularize.
MP#2: Pick and Pray
  • Goal: a correlation between number of colonies and transformation success was seen in our project. Those constructs with zero or little colonies (less than 30) were assigned to the MP1, while those that had a high number of colonies were screened again by selecting more colonies (short protein 10).
  • Dates: 15/07/2024
  • Results: we did not get any results. The project ended up stopping since we had mutations in the short peptides and the effort to fix them was not worth it.
MP#3: Spearhead
  • Goal: The cloning of the linker was of great importance because it allowed for the cloning of gblocks that we had received by that time. The goal was to prioritize the process of cloning for the linker, following the same steps as in the MP1.
  • Dates: 15/07/2024 -> 16/07/2024
  • Results: The linker was cloned successfully but after Sanger sequencing analysis we discovered some mutations in the sequence that lead to a frame shift and ultimately made it useless.
MP#3.5: Spearhead.5
  • Goal: The goal was to rapidly clone the gblock modA (that binds Mo), as the drylab team could use the data generated in the quantification to perform models and enhance the activity of modA.
  • Dates: 16/07/2024 -> 30/07/2024
  • Results: Positive results showing the correct insertion of the gBlock were seen after Sanger Sequencing. However, the linker was still mutated even after using a new primer set for amplification, so the sequences needed to be fixed by (The first time all the obtained colonies were negative).
MP#3.9: Spearhead.9
  • Goal: ModA turned out to be not as easy to modulate because of the lack of tools to effectively predict Mo ions binding. Instead, HRMBO, Landmoduling and SilE were prioritized, as Ni and lanthanides seemed to fit our expertise better.
  • Dates: 29/07/2024 -> 02/08/2024
  • Results: HRMBP, Landmodulin, and SilE were introduced into the gBlock protein library. A miniprep for gblocks 13.4, 14.4, 15.4 (HRMBP, Lanmodulin, SilE) and sending for Sanger sequencing, where we achieved a positive construct of SilE and HRMBP. Landmodulin was discarded after this.
MP#4: Show must go on
  • Goal: The goal of this miniproject was to continue with the construct that was detected via PCR and prepare O/N cultures for performing a miniprep and sending the plasmids to sequence.
  • Dates: 15/07/2024 -> 22/07/2024
  • Results: Sanger sequencing showed some mutations for the successful inversed PCR of the short peptides. A new strategy for the cloning of these needed to be performed. A few of these proteins did not have any mutations. These were used for the preparation of O/N culture after checking in arabinose plates if there was any GFP presence. The O/N cultures were used for the preparation of glycerol stock on the next day.
MP#5: Is MBP (p)BAD for you?
  • Goal: The goal was to substitute sfGFP for MBP upstream, the cloning site of the short peptides and gblocks. To do so an overhang PCR was performed for the MBP sequence followed by Gibson Assembly of the MBP into the sdGFP p BAD vector.
  • Dates: 15/07/2024 -> 19/07/2024
  • Results: MBP was correctly inserted into the pBAD vector and substituted GFP, as we could see by colony PCR, sanger sequencing and arabinose induction (no GFP induction was seen).
MP#6: Behead Bsal
  • Goal: Once MBP was cloned into pBAD, a BsaI cut site was detected inside the MBP sequence. To eliminate it, a SDM (Side Directed Mutagenesis) PCR was performed. After transformation, an endonuclease assay was performed using the original MBP pBAD sequence and comparing it to the BsaI mutated cut site.
  • Dates: 19/07/2024 -> 30/07/2024
  • Results: Three of the four samples successfully removed the Bsal cut site, as we showed in the next assay.
BsaI restriction
Figure 1: Restriction with BsaI, project MP#6. The last three sample do not present a BsaI so the SDM worked
MP#7 and MP#10: A new horizon
  • Goal: Once MBP was free of internal BsaI cut sites, it was ready to be implemented into our system for protein expression. The positive results obtained through the Sanger sequencing on MP#4 could be amplified using overhang PCR and introduced by Gibson Assembly into the MBP pBAD vector.
  • Dates: 25/07/2024 -> 22/08/2024
  • Results: Although the MP was started early and the first PCR to introduce overhangs for the Gibson Assembly was already done, we decided that there was no point in doing this project, as our solution was now centered on in-vivo remediation (using the cells) instead of using purified proteins, which was the main point of using MBP as the carrier for our protein.
MP#8: Catch me if you can
  • Goal: The gBlocks containing our SpyTag system finally arrived, and we started the process of cloning into pBAD and pBAD2. PBAD2 was supposed to be a plasmid with a compatible ORI and a different selection marker (cm) than pBAD (kan). The idea was to co-transform a culture so it could produce both monomers required for the polymerization of the system.
  • Dates: 17/07/2024 -> 05/09/2024
  • Results: Although we had some difficulties with this project, we were able to clone into pBAD. Unfortunately, we had to drop pBAD2 since it was not working.
MP#9: I can fix him
  • Goal: The sanger sequencing from MP#1 and MP#3 indicated the presence of variable mutations on the short peptides (those introduced by inverse PCR). The goal of the project was to use new specific primers designed for SDM for each of the proteins to “fix them”.
  • Dates: 29/07/2024 -> 14/08/2024
  • Results: Several strategies were used to perform this SDM, the final result was overall positive, as most of the proteins were successfully cloned into the pBAD plasmid.
MP#9.5: I can fix him.5
  • Goal: After the cloning of the linker downstream sfGFP in pBAD, a deletion was detected upon receiving Sanger sequencing results. This deletion translated into a frameshift that made it impossible for the expression of the gBlocks proteins cloned downstream of the linker. The goal here was to use a SDM primer set to fix each of the gBlocks.
  • Dates: 1/08/2024 -> 16/08/2024
  • Results: Several strategies were used to perform this SDM, the final result was overall positive, as most of the proteins were successfully cloned into the pBAD plasmid.
MP#11: Please work
  • Goal: Development and standardization of a toxicity assay for different types of metals and concentrations. The idea was to use it as a screening platform that could be adapted to our protein library.
  • Dates: 22/07/2024 -> 05/09/2024
  • Results: The toxicity assay left a bitter taste, as a lot of days and effort were put into this. However, it was only at the end of the project that the results started to make sense and be reproducible. Overall, this MP showed the toxic ranges for E. coli of the metals we were working with.
MP#12: Golden gate for Short peptides
  • Goal: New strategy for cloning short peptides that could not be cloned or fixed beforehand. It was based on available online information and PI counseling. The idea was to include overhangs that codify for BsaI cut sites in both flanks of the inverse PCR part, with the intention of digesting them after PCR and using the overhangs as anchoring points for a more efficient ligation, hoping that the mutations in the middle of the sequence would stop.
  • Dates: 25/07/2024 -> 19/08/2024
  • Results: The results of this new strategy were overall good, as all short peptides that could not be cloned during MP1 or MP4 were now cloned and expressed successfully.
MP#13: Piggy bank
  • Goal: Small MP with the aim of preparing and labeling glycerol stocks for the high number of positive constructs we were achieving by that point. Glycerol stocks allowed an easy way of preparing standardized O/N cultures.
  • Dates: 06/08/2024 -> 08/08/2024
  • Results: The glycerol stocks were successfully prepared and labeled.
MP#14: Safety form
  • Goal: Small MP with the aim of completing the iGEM deliverable for the safety form.
  • Dates: 07/08/2024
  • Results: The safety form was completed and submitted on time.
MP#15: Positive in Tag
  • Goal: The aim was to clone the positive results (both gBlocks and short peptides) into the tag sequence (by substituting sfGFP for the coding sequence of the metal binding protein). This would be done by classic restriction enzyme cloning.
  • Dates: 12/08/2024 -> 26/08/2024
  • Results: This project was put on pause due to the cloning of the catcher taking longer than expected. Only a few constructs were inserted into the tag sequence.
MP#16: Glycerol stock
  • Goal: Small MP with the aim of preparing and labeling glycerol stocks. Glyerol stock (P1, J140,MymT,PdBP, GolB, MT_SYNSP, MT, ArsR, Azurin, Csp1, HRMBP, SilE, ModA).
  • Dates: 20/08/2024
  • Results: The glycerol stocks were successfully prepared and labeled.
MP#17: Third chance for the gBloks
  • Goal: Cloning some gBlocks that could not be fixed by SDM in MP 9.5. To do so, the correct linker sequence (fixed) was used this time. The cloning protocol was a classic Golden Gate and was used for Csp1 and for the gBlocks received by the dry lab, with mutated sequences that could enhance the activity of the protein. These were Csp1.3, Csp1.8 and SmtB.7.
  • Dates: 19/08/2024 -> 04/09/2024
  • Results: The gBlocks were easily cloned using the golden gate strategy by using the linker sequence from an already corrected protein. The drylab sequences were correctly cloned after a second attempt (the first time all of the cloned sequences corresponded to the ModA sequence, indicating a possible mixture of labels or tubes).
MP#18: Cool plates
  • Goal: A new screening platform needed to be implemented to test for the metal binding capabilities of our protein library because the toxicity assay was not completely matured by this point. The goal was to develop and standardize a protocol for creating agar plates with a gradient of metal concentration.
  • Dates: 20/08/2024 -> 09/09/2024
  • Results: We developed a new method that can cherry-pick the fittest bacterial strain against a certain metal concentration. The agar plates preparation was standardized, and we switched to spot-assay to improve results visualization.
MP#19: Colorimetric assay
  • Goal: In our pursuit of measurement mention, we aimed to develop a high throughput method for Copper detection based on colorimetric approaches. This method was meant to quantify the Copper recuperation from a lab water sample containing high levels of Cu on it, and use it as benchmark for the different proteins of the library.
  • Dates: 20/08/2024 -> 09/09/2024
  • Results: We adapted a previously reported assay from the literature as an alternative to purchasing a commercial kit, successfully establishing linearity in our standard curve for the concentration range of interest. Although we obtained valuable results, there were challenges during the assay preparation that need further optimization to improve its robustness.

During our wet lab work, we used Benchling’s digital logbook as our primary tool for documentation. In addition to this digital notebook, we maintained a physical one, where we mainly added sequencing labels to keep track of all the proteins we sent for sequencing. To complement these, we also used a shared folder on Drive to organize and store photos of the PCR gels, toxicity assays, colorimetry results and other data that is too complex to include in the digital notebook.

You can explore the referenced digital notebook below:

Dowload the pdf here if it does not show in your browser

Drylab Notebook

Like in wet lab our work was organized between mini-projects, with each project being distributed throughout the dry lab team. These projects were organized in our dry lab notebook where our results and other information were noted down. On this page, you can find the notebook and description of each mini-project.

MP#1: Protein Spearhead Choice
  • Goal: Decide on which protein will be used for further experiments.
  • Dates: 08/07/2024 -> 14/07/2024
  • Results: Based on evolutionary analysis, structure prediction, and metal availability, Csp1 and SmtB were chosen as the target proteins.
MP#2: Binding Site Prediction
  • Goal: Establish baseline data for metal binding in Csp1 and SmtB.
  • Dates: 15/07/2024 -> 23/07/2024
  • Results: Identified the key residues involved in metal binding and simulated docking within the 3-dimensional protein structure.
MP#3: Machine Learning Model
  • Goal: Identify metal-binding features through machine learning.
  • Dates: 06/08/2024 -> 10/08/2024
  • Results: The neural network was constructed but the lack of conclusive data from toxicity assays affected the development of an adequate training data set to pursue further results.
MP#4: Molecular Dynamics
  • Goal: Understand the physics behind metal binding in our proteins.
  • Dates: 06/08/2024 -> 16/09/2024
  • Results: Modeling metal in forcefields was more complex than anticipated as they form non-covalent bonds, so our simulations did not provide concrete results.
MP#5: Binding Optimization
  • Goal: Optimize metal binding from the wild-type sequences for Csp1 and SmtB.
  • Dates: 24/07/2024 -> 16/09/2024
  • Results: Through metal binding analysis software, our mutated sequences (specifically Csp1 #3, 7, and SmtB #8) were seen to show improved binding to their respective metals. These sequences were then sent to wet lab to use as potential enzymes with improved metal-binding capacity.

During our dry lab work, we used Benchling’s digital logbook as our primary tool for documentation. In addition to this digital notebook, we used online browser interfaces of published, open-source metal-binding software (either hosted by the publisher’s domain or ported to Hugging Face, a collaborative machine-learning community).

You can explore the referenced digital notebook below:

Dowload the pdf here if it does not show in your browser