iGEM 2022 — Team tuebingen

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}What was done when?{% endblock %}
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#### Week 1▼

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#### Week 2▼

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#### Week 3▼

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* 17.1.2022: kickoff meeting for iGEM Team Tuebingen 2022

#### Week 4▼

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* 24.1.2022: weekly team meeting

#### Week 5▼

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* 31.1.2022: weekly team meeting

#### Week 6▼

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* 7.2.2022: weekly team meeting

#### Week 7▼

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* 14.2.2022: weekly team meeting
* 20.2.2022: teambuilding: first in-person meeting at the castle in Tuebingen

#### Week 8▼

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* 21.2.2022: weekly team meeting
* 25.2.2022: finance intro meeting with members from old team
* 26.2.2022: project finding day – decision to further research 3 ideas
* 27.2.2022: logo version 1 finished

#### Week 9▼

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* 28.2.2022: weekly team meeting
* 6.3.2022: team building: hiking trip

#### Week 10▼

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* 7.3.2022: weekly team meeting, final logo finished
* 13.3.2022: project discussion day - decision to further pursue creating a biosensor for Anatoxin-a

#### Week 11▼

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* 14.3.2022: weekly team meeting, implementation of final logo

#### Week 12▼

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* 21.3.2022: weekly team meeting
* 25.3.2022: journal club: presenting key papers on Anatoxin-a and biosensors
* 26.3.2022: drylab meeting
* 27.3.2022: Human Practices meeting

#### Week 13▼

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* 28.3.2022: weekly team meeting; SnapGene sponsorship
* 29.3.2022: wetlab team meeting
* 1.4.2022: meeting with Libera
* 2.4.2022: first meeting of website subteam
* 3.4.2022: Human Practices meeting

#### Week 14▼

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* 4.4.2022: weekly team meeting
* 7.4.2022: finance meeting with ThermoFisher

#### Week 15▼

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* 11.4.2022: weekly team meeting
* 12.4.2022: Carl Roth sponsorship
* 14.4.2022: full website is online

#### Week 16▼

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* 18.4.2022: weekly team meeting, meeting with iGEM Team Heidelberg
* 20.4.2022: finance meeting with Brand, finance meeting with mentors
* 21.4.2022: finance meeting with DS Stiftung
* 22.4.2022: iGEM registration fee payment
* 24.4.2022: Human Practices meeting

#### Week 17▼

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* 25.4.2022: weekly team meeting
* 26.4.2022: Microsynth sponsorhsip, Drylab Meeting: AutoDock Vina

#### Week 18▼

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* 2.5.2022: weekly team meeting, final version of portfolio

#### Week 19▼

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* 9.5.2022: weekly team meeting
* 10.5.2022: interview with cyanobacteria and cyanotoxin expert
* 11.5.2022: meeting with Dr. Forchhammer

#### Week 20▼

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* 16.5.2022: weekly team meeting
* 17.5.2022: drylab meeting: auto dock
* 21.5.2022: designed and printed business cards

#### Week 21▼

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* 23.5.2022: weekly team meeting, Azenta Life Science Sponsorship, Promega grand awarded
* 26.5.2022: meeting with Wency
* 27.5.2022: team building: dinner

#### Week 22▼

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* 30.5.2022: weekly team meeting
* 1.6.2022: Drylab: Detecting Binding Sites with SSnet
* 3.6.2022: finance team meeting

#### Week 23▼

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* 6.6.2022: weekly team meeting
* 7.6.2022: finance meeting with CodexDNA, meeting ith Khaleb
* 8.6.2022: meeting with Victor Sourjik

#### Week 24▼

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* 13.6.2022: weekly team meeting
* 19.6.2022: meeting with Wency

#### Week 25▼

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* 20.6.2022: weekly team meeting
* 22.6.2022: safety introduction in the Forchhammer lab
* 24.6.2022: CMFI financial support
* 26.6.2022: Human Practices meeting

#### Week 26▼

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* 27.6.2022: weekly team meeting, NEB sponsorship, DWK sponsorship
* 28.6.2022: Greiner-Bio-One sponsorship, Nippon Genetics Europe sponsorship, meeting with Dr. Joachim Schultz
* 29.6.2022: joined lab meeting in the Forchhammer lab
* 1.7.2022: Hamburg European Meetup, IDT sponsorship
* 2.7.2022: Hamburg European Meetup
* 3.7.2022: Hamburg European Meetup

#### Week 27▼

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* 4.7.2022: weekly team meeting
* 7.7.2022: finance team meeting
* 10.7.2022: Human Practices meeting

#### Week 28▼

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* 11.7.2022: weekly team meeting, first day in the lab: set up lab, start culturing PctD-LBD strain
* 12.7.2022: Wetlab: isolation of PctD-LBD plasmid
* 13.7.2022: Drylab: Molecular Docking with SeeSar
* 14.7.2022: Impact Grant form submitted
* 15.7.2022: Wetlab: transformation of BL21 DE3 plysS cells with PctD-LBD plasmid

#### Week 29▼

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* 18.7.2022: weekly team meeting website subteam meeting, wetlab: autoclave material, meeting with Dr. Oesterheld
* 19.7.2022: Drylab: meeting with Team from Kopenhagen, Wetlab: colony PCR of PctD-LD transformed strain, preculture for PctD-LBD expression
* 20.7.2022: Wetlab: prepare buffers for purification, expression of PctD-LBD and harvesting of cells
* 21.7.2022: MedChemExpress sponsorship, Wetlab: PctD-LBD purification failed

#### Week 30▼

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* 25.7.2022: weekly team meeting
* 27.7.2022: meeting with MedChemExpress, Drylab: meeting with Prof. Thiel
* 28.7.2022: Wetlab: propagation of competent NEB5alpha cells for interlab study
* 29.7.2022: Wetlab: redo propagation of competent NEB5alpha cells for interlab study
* 30.7.2022: video: First meeting, discussion of equipment, roles, schedule
* 31.7.2022: Impact grand awarded, Human Practices meeting

#### Week 31▼

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* 1.8.2022: weekly team meeting, Wetlab: interlab study: culture NEB5alpha cells and calibrate plate reader; PctD-LBD expression: SDS gel to check expression; start generating competent VS994 cells
* 2.8.2022: Wetlab: interlab study: continue preparation of competent cells, prepare buffers for purification
* 3.8.2022: Wetlab: agarose gel for colony PCRs
* 4.8.2022: Wetlab: interlab study: check transformation efficiency of competent cells; purification of D-PctD seems successful
* 5.8.2022: Wetlab: transformation of competent cells failed
* 6.8.2022: video: Meeting with Dr. Khaled Salim to discussion of venues for shooting and shooting in the lab

#### Week 32▼

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* 8.8.2022: weekly team meeting
* 9.8.2022: Wetlab: SDS page of PctD-LBD purification, agarose gel of PCRs, interlab study: transformation with interlab constructs
* 10.8.2022: Wetlab: interlab study: transformation failed, redid with purchased NEB5alpha competent cells, video: Meeting of the whole team to take team drone shots
* 11.8.2022: Wetlab: interlab study: transformation of constructs 1,2,3 successful -> continued incubation; preparation of competent cells continued, plasmid isolation of VS1001, preculture of VS1007 failed
* 12.8.2022: Wetlab: meassure concentration of purified PctD-LBD -> total yield of app. 1.75mg protein, interlab study: after 40h of incubation colonies for all constructs are present, Human Practices meeting
* 14.8.2022: Human Practices meeting

#### Week 33▼

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* 15.8.2022: weekly team meeting, Wetlab: calibration for interlab study, video: Dicussion of differents variants of text for the script; discussion which shots to use for which part of text, discussion of animation for parts of our video
* 16.8.2022: Wiki/Design meeting, wetlab: interlab study. Redo transformations with constructs; creation of chimeric receptor: transformation of NEB5alpha with pKG116 plasmid
* 17.8.2022: Wetlab: interlab study: “old” colonies fluoresce under microscope -> use for further experiment, new plates have no colonies -> incubate further; Creation of chimeric receptor: grow cells for plasmid prep of pKG116; gradient PCR of EnvZ and EnvZ-HAMP shows corect amplification of EnvZ-HAMP but not EnvZ
* 18.8.2022: Wetlab: interlab study: meassurment of constructs; PCR of PctD, PctD-HAMP and pKG116 shows correct amplification of PctD and PctD-HAMP but not pKG116
* 19.8.2022: Wetlab: interlab: repeat meassurment of constructs; gradient PCR of EnvZ and pKG116

#### Week 34▼

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* 22.8.2022: weekly team meeting, Launch overhauled website, Wetlab: preculture for new PctD-LBD expression, gradient PCR of EnvZ failed, video: Filming of cyanobacteria in culture and under the microscope in the laboratory; final script discussion (camera shots, text, ...)
* 23.8.2022: Gibson Assembly Kit bought, Wetlab: main culture and harvesting of cells for PctD-LBD purification, EnvZ gradient PCR with Q5 polymerase works, video: Shot sequence of talking person in the lab (Part "Deep project dive")
* 24.8.2022: Drylab: Meeting with team from Greece, visiting the central public health authority of Baden-Württemberg
* 25.8.2022: Wetlab: purification of PctD-LBD shows 2 peaks that could contain PctD-LBD
* 26.8.2022: video: Final script corrections, choosing of background speaker, discussion of questions regarding animation, editing, cutting, subtitles, attributions

#### Week 35▼

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* 29.8.2022: weekly team meeting, Wetlab: SDS page of PctD-LBD purification -> protein contained in one peak, fraction pure, dyalisis of protein fractions against ITC buffer, chimeric chemoreceptor generation: purification of PCR products and Gibson Assembly, transformation of assembly product
* 30.8.2022: Wetlab: PctD purification: meassure protein concentration, chimeric chemoreceptor generation: transferred 20 colonies per construct (1 and 2) on new plates
* 31.8.2022: Wetlab: chemoreceptor generation: colony PCR of 20 colonies per construct
  Video: Final discussion about editing, subtitles, attributions
* 1.9.2022: Wetlab: chemosensor generation: agarose gel of colony PCR shows positive control for most colonies of contruct 2 -> overnight culture of positive colonies, meeting with Libera
* 2.9.2022: Wetlab: chemoreceptor generation: plasmid isolation of selected PCR-positive colonies, video: Uploaded final version

#### Week 36▼

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* 5.9.2022: weekly team meeting
* 6.9.2022: Wetlab: sent plasmids to sequencing, started propagation of cells (VS996, VS1001) for chemically competent E.coli, Drylab: meeting with team Patras
* 7.9.2022: Wetlab: continuation of preparation of chemically competent E.coli
* 8.9.2022: Wetlab: continuation of preparation of chemically competent E.coli, test transformations with pUC19, received sequencing results, Human Practices survey in Tuebingen

#### Week 37▼

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* 12.9.2022: weekly team meeting, Wetlab: continuation of transformation of VS996 and VS1001 with pUC19 and VS1008 and pUC19 and PctD-HAMP-EnvZ respectively
* 13.9.2022: Wetlab: checking results of transformations, plasmid isolation of VS1001 and sending for sequencing, Gibson assembly of PctD-EnvZ-Hamp and PctD-Hamp-EnvZ and transformation into NEB5alpha competent cells
* 14.9.2022: Wetlab: preparation of chemically competent VS1007 cells, colony PCR and re-streaking of transformed cells from the day before
* 15.9.2022: Wetlab: gel electrophoresis of colony PCR, preparation of Choline chloride stock for ITC measurement
* 16.9.2022: Wetlab: ITC meassurement of PctD-LBD, plasmid isolation of transformed cells and sending for sequencing

#### Week 38▼

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* 19.9.2022: Wetlab: received sequencing results, transformation of the hybrid receptor constructs (PctD-EnvZ-Hamp and PctD-Hamp-EnvZ)
* 20.9.2022: Wetlab: checking transformation results
* 21.9.2022: Wetlab: subculturing of VS1001 PHE
* 22.9.2022: Wetlab: inoculating overnight cultures of VS1001 PEH, VS1001 PHE, VS1007 PEH, PCR, SDS Gel
* 23.9.2022: Wetlab: inducing of cultures with salicylic acid

#### Week 39▼

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* 26.9.2022: weekly team meeting, Wetlab: received sequencing results
* 27.9.2022: Wetlab: measuring bacterial growth curves (VS996, VS1001, VS1007, VS1001+PEH, VS1001+PHE, VS1007+PEH, VS1007+PHE)
* 28.9.2022: Wetlab: SDS Page of VS1001 PEH, VS1001 PHE and VS1007 PEH, Wiki: Planning meeting and first implementations
* 29.9.2022: Wetlab: test of GFP expression of the biosensor: fluorescence measurement of VS1001, VS1001+PEH, VS1001+PHE, VS1007, VS1007+PEH, VS1007+PHE after induction with Aspirin, Human practices: Visit to the chemical testing facility CVUA Sigmaringen
* 30.9.2022: Wetlab: repeated test of GFP expression of the biosensor, Wiki: implementation session
* 2.10.2022: Wiki: discuss further To-Dos

#### Week 40▼

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* 3.10.2022: weekly team meeting
* 4.10.2022: Wetlab: resistance testing of VS1007, preparation of glycerol stock of VS1007-PEH
* 5.10.2022: Wetlab: checking resistance test of VS1007, continuation of preparation of glycerol stock of VS1007-PEH, preparation of stock solutions for acetylcholine, choline, acetylsalicylic acid, prepared quantitative testing of biosensor VS1007-PEH
* 6.10.2022: Wetlab: continuation of testing of biosensor VS1007-PEH failed (no bacterial growth), troubleshooting for testing of biosensor, prepare new testing of biosensor
* 7.10.2022: Wetlab: quantitative testing of biosensor VS1007-PEH with plate reader over period of 4h
* 9.10.2022: Wiki: implementation session

#### Week 41▼

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* 10.10.2022: weekly team meeting
* 12.10.2022: Wiki: implementation session
* 13.10.2022: Wiki: review session

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}The goal we set for ourselves to create a biosensor for Anatoxin-a required clean execution of all steps of the design cycle.{% endblock %}
{% block page\_content %}

## Design

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When we first decided to pursue the Anatoxin-a biosensor project, we envisioned to combine a part of a receptor that can bind Anatoxin-a with an intracellular signalling system in *E. coli* to result in an expression of a reporter protein. Since Anatoxin-a strongly and irreversibly binds nicotinergic acetlycholine receptors (nAchRs), we first anticipated to employ the soluble homologue, namely acetylcholine binding protein (AChBP). Even so, AchBPs are usually expressed by eukaryotic systems, there is evidence that it can also be efficiently expressed in *E. coli* [(2)](#2).

However, after literature review and discussion with our Principal Investigator Libera lo Presti we decided AChBP would not be suitable for the use in our biosensor because of its pentameric structure and an unusual way of signal transduction compared to bacterial sensing systems.

Instead, we chose to employ the recently characterized PctD chemoreceptor that shows acetylcholine detection for ligand binding[(3)](#3). We contacted Victor Sourjik (MPI Marburg), an expert in the field of creation of hybrid receptors, to advise us on the design of our receptor hybrid. He and Wenhao Xu, a postdoc in his group, confirmed that a combination between PctD and EnvZ, a histidine kinase part of bacterial two-component signalling systems, could be feasible. They offered us templates for PctD, as well as a reporter plasmid that expresses GFP in response to EnvZ activation.

We then approached Joachim Schultz (University of Tuebingen) to help us with some details of the fusion between PctD and EnvZ. We designed two versions of the chimeric receptor differing in the fusion site between the two receptors. Cells carrying the reporter plasmid, as well as these chimeric receptors in another plasmid, were envisioned to induce the expression of GFP upon ligand binding to PctD.

## Build

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The first step to be performed at the building stage in the wetlab was to assemble our selected hybrid receptors in DH5a cells. For this cloning step, we decided to use Gibson Assembly for its ability to produce scar-free fusion proteins, critical in our case, since the fusion point is crucial for the hybrid receptor functioning. Then, we amplified the different linear fragments via PCR that later were used for the Gibson Assembly and transformation in DH5a cells. Later on, to finish our device we transformed the correct hybrid receptor into the reporter strain VS1007 harbouring a reporter plasmid with GFP expression under the promoter OmpC. The chemoreceptors and EnvZ genes were deleted previously from this strain.

## Test - Prediction of Anatoxin-a binding to PctD

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The main goal we set in the drylab was to test the binding of Anatoxin-a to PctD. Besides, we wanted to predict potential mutations in the ligand binding domain that enhance the binding. We obtained the experimentally determined structure of PctD, the chemical structure of Anatoxin-a, the natural ligands acetylcholine, choline and betaine to compare predicted binding.

First we tested the potential binding site of Anatoxin-a to PctD via SSnet [(4).](#4)  To verify the results obtained from SSnet, we employed SeeSar [(5)](#5) which ouputs predictions of affinities for potential binding sites.

Finally,we collaborated with iGEM Team  [iGEM Team Patras](https://2022.igem.wiki/patras/)  to utilize Gromacs [(6)](#6) for Molecular Dynamics Simulation to predict binding while accounting for dynamic changes in the receptor structure.

## Test - Anatoxin-a binding to PctD-LBD

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Despite the unpromising results from drylab, we still wanted to test Anatoxin-a binding to PctD  *in vitro*  as well. The interaction characterization between PctD and Anatoxin A is a quantitative result that would determine whether our idea would fulfil the original purpose of measuring this harmful cyanotoxin. To do this, we had planned a Isothermal Titration Calorimetry Experiment with the natural ligands Acetylcholine and Choline as controls, previously performed for the characterization of PctD by Dr. Tino Krell, and Anatoxin-a. The first step for this experiment was the expression of the PctD-Ligand Binding Domain (LBD), in *E. coli* , that was purified via an Immobilized Metal Affinity Column (IMAC). The purification was successful and tested through with an SDS-PAGE gel. Then, we measured the binding of the PctD-LBD with the natural ligands previously mentioned in the microcalorimeter device.

## Test - Biosensor construction

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During the construction of our biosensor plasmids, the first test was getting the correct PCR fragments for the Gibson Assembly, which took some troubles due to the overlapping regions required for this cloning technique. After the transformation, the screening for the correct hybrid receptor construct was performed with colony PCR to see the length of the 2 fragments assembled with the backbone vector. Within the positive colonies, a representative number of isolated plasmid samples were sent to Sanger sequencing to confirm the right sequence.

For our engineered strain containing the reporter plasmid and the hybrid receptor expression plasmid, we tested different three conditions compared to the strain with only the reporter plasmid. First, we tested the induction with acetylsalicylic acid to produce the hybrid receptor at 2 µM and 4 µM, which was not noticeable in a SDS-PAGE where we analysed the cell extracts after 0, 2 and 6 hours, this being consistent with previous experiments by other iGEM teams with this promoter in a mid-low copy number plasmid. Second, we checked at the fluorescence microscope the presence of GFP expresion. To our surprise, the strain harbouring the reporter plasmid VS1007, derived from pUA66, showed fluorescence despite being under the regulation of the inducible promoter OmpC, as well as the strain containing the hybrid receptor with and without the ligand present in the media. Showing us a possible leakiness of the OmpC promotor. Third and foremost, with the intention to test the final application of our engineered strain, we performed a plate reader experiment where we tested different concentrations of the natural ligands during 4 hours in minimal media. The testing with Anatoxin-a was not possible since the provider had issues for the production, acquisition and delivery of the product, which they informed us on a short notice that they would no longer provide the compound.

## Learn

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For the learning stage, several setbacks pushed us back and taught us what to improve. First, the planification of experiments, including member or material availability and documentation plays a crucial role in the success of an experiment. Another underestimation we did was setting up the lab, including the material organization or antibiotics stocks, which are essential for the appropriate selection of the plasmids and strains. These together with a small team working part time, we learnt that everything takes longer than originally planned.

One of the wetlab experiments we planned and executed was the purification of PctD and the ITC measurement. The results we got from ITC suggest that, even though we successfully purified PctD, most of the protein is not functional. This means that we need to verify that our protein is denatured or misfolded and then evaluate and redesign our purification for future experiments and design measures to refold the protein, if needed. It also made us think about the importance of checking the protein functionality before conducting further experiments like ITC.

Another issue we had with the ITC measurement and also our biosensor testing was the fact that we were not able to get Anatoxin-A on time, because our deliverer postponed the delivery and then cancelled in on short-notice. From this we learned that it is important to have multiple sources for crucial compounds and that we act earlier when faced with delivery issues in the future.

One of the biggest issues we had to deal with in our initial experiments was the amplification of the DNA fragments by PCR. At the beginning we used the pfu DNA polymerase in all our PCR reactions, but it turned out that it only worked for some of them. We thought that the issue could come from the annealing temperature of the primers, so we did a gradient PCR to cover a wide range of annealing temperature as well as added Q5 GC enhancer buffer, but it was not successful either. Surprisingly, by using the Taq polymerase all the PCRs were successful, but due to its high rate of error we decided to not use it for our experiment. After searching in the literature and consulting with members of the lab, we finally decided to use the Q5 DNA polymerase for our PCRs, and it was a success as all the PCR reactions worked from the first time. From this we have learned that PCR troubleshooting is an important part to know and probably we will have to deal with it in the future.

At advanced stages of our experiments, we found out that our chimeric receptor has an unexpected point mutation close to the binding pocket that might affect to the affinity and specificity. This was really unfortunate as the mutation was already existing in the pctd sequence that we used as template, but we did not realise in the beginning. Therefore, we have learned from this experience that checking the starting material in the beginning of a project is a crucial step to do because otherwise you might find some unexpected events at later stages that could break all your plans.

Upon testing of our biosensor qualitatively and quantitatively we found that our reporter plasmid introduces some background fluorescence, and that our biosensor candidate VS1007 PEH shows high fluorescence without ligands present. But we could not observe a clear change in fluorescence when acetylcholine or choline where added, therefore not verifying any sensing of these substrates. This indicates that our chimeric receptor might not work. The testing of the biosensor therefore needs to be redone to verify whether the substrates have no effect on the biosensor. Then we need to start another engineering cycle.

Drylab results predicting the binding of Anatoxin-a to PctD were also not promising. In all three used tools, Anatoxin-a binding was considerably weaker at the acetlycholine binding site or the optimal binding site was predicted in the intracellular part of the receptor, which in our hybrid receptor design is replaced by EnvZ.

When starting another engineering cycle and re-designing our chimeric receptor, one way to do it is by prediction of structural changes to the ligand binding site affecting the structure in the wetlab. Initially, the plan was to use the drylab results to predict how binding of Anatoxin-a to PctD could be improved. However, we learned talking to Prof. Thiel, that prediction of effects of mutations on binding is difficult to conduct reliably. Therefore, we would need to shift this part entirely to wetlab, by having a lybrary of different variants of the chimeric receptor synthesized which could then be screened.

Other steps we want to take in our re-designed project plan is to generate primers to use PCR to remove of the mutation in our current “PEH” chimeric receptor and also reassess the expression and purification of our PctD-LBD to redo the binding study of acetylcholine and choline to the protein. When this has been successful, testing with Anatoxin-a is the next step.

Overall, when looking back at our project, we can say that we gained a lot of insights into the different stages of engineering, that we could use to improve our project and future projects. We learned that establishing methods for building and testing a biosensor takes a lot of time and that controls are crucial on every step of the process.

## References

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1. Matilla MA, Velando F, Tajuelo A, Martín-Mora D, Xu W, Sourjik V, Gavira JA, Krell T. Chemotaxis of the Human Pathogen Pseudomonas aeruginosa to the Neurotransmitter Acetylcholine. mBio. 2022 Apr 26;13(2):e0345821
2. Abraham N, Paul B, Ragnarsson L, LewisRJ (2016)Escherichia coliProtein ExpressionSystem for Acetylcholine Binding Proteins (AChBPs).PLoS ONE 11(6): e0157363
3. Matilla MA, Velando F, Tajuelo A, Martín-Mora D, Xu W, Sourjik V, Gavira JA, Krell T. Chemotaxis of the Human Pathogen Pseudomonas aeruginosa to the Neurotransmitter Acetylcholine. mBio. 2022 Apr 26;13(2):e0345821
4. Verma N, Qu X, Trozzi F, Elsaied M, Karki N, Tao Y, Zoltowski B, Larson EC, Kraka E. SSnet: A Deep Learning Approach for Protein-Ligand Interaction Prediction. Int J Mol Sci. 2021 Jan 30;22(3):1392. doi: 10.3390/ijms22031392.
5. SeeSAR version 12.1.0; BioSolveIT GmbH, Sankt Augustin, Germany, 2022, www.biosolveit.de/SeeSAR
6. H. Bekker, H.J.C. Berendsen, E.J. Dijkstra, S. Achterop, R. van Drunen, D. van der Spoel, A. Sijbers, and H. Keegstra et al., “Gromacs: A parallel computer for molecular dynamics simulations”; pp. 252–256 in Physics computing 92. Edited by R.A. de Groot and J. Nadrchal. World Scientific, Singapore, 1993.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}All the experiments we conducted{% endblock %}
{% block page\_content %}

## Wetlab

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### General Protocols --- Agarose Gel Electrophoresis▼ Introduction The agarose gel needs to be prepared for the future electrophoresis. Materials * 0.6 g Agarose * 60 ml 1x TAE buffer * Midori Green DNA Dye * 250 ml Erlenmeyer flask * 100 ml measuring cylinder * Scale * Weighing spoon * Agarose electrophoresis chamber (BioRad) * Power output for electrophoresis Procedure * Measure 60 ml of 1x TAE buffer and pour it in the Erlenmeyer flask * Add 0,6 g of Agarose to the solution * Microwave until the solution is clear, do not overboil * Before adding the Midori Green Dye make sure, the solution is not hot. Cool it down by pouring water on the outside of the Erlenmeyer flask with water in the sink * Midori Green Dye is potentially carcinogenic, handle it cautiously, wear gloves and a mask. * Add 2µl of Midori Green Dye * Mix by swirling the Erlenmeyer flask around 5-10 times * Pour agarose gel and let set until solid * Load gel and let run --- SDS PAGE▼ --- Materials * pre-made 10% SDS gel * SERVA Triple Color Protein Standard III * 1x SDS runnning buffer * electrophoresis chamber * Coomassie Staining colution ("Der Blaue Jonas", GPR) Procedure * add 2x lämmli buffer to sample * heat 95°C for 5-10 min * load gels and run at 200V for 45min * rinsed gel with VE water, then incubated 10 min with Coomassie Staining colution ("Der Blaue Jonas", GPR) while shaking manually * rinsed the gel twice with VE water * scan image Plasmid Isolation (commercial kit)▼ --- Introduction Plasmid Isolation is a crucial step for the future procedures like cloning, DNA sequencing etc. The aim is to purify the plasmid from the bacteria by removing all the cell components, proteins and chromosomal DNA and RNA molecules. Materials * Bacteria culture * 5 ml LB medium * (1 x 1000)V Antibiotic * 250 μl P1 Buffer (resuspension buffer) * 250 μl P2 Buffer (lysis buffer) * 350 μl N3 Buffer * 0.5 ml PB Buffer * 0.75 ml Buffer PE * 50 μl Nuclease free water * QIAprep 2.0 spin column * Microcentrifuge Tube Procedure * Add 1-5ml LB medium to autoclaved glass tubes. * Add antibiotic corresponding to the resistance of the plasmid that needs to be isolated * With a pipette tip, take some bacteria from a plate and inoculate the LB medium * Incubate overnight (12-15h) at 120-150 rpm, 37°C, roughly 45° angle * Pellet 1-5 ml bacterial overnight culture by centrifugation at >8000rpm (6800 \* g) for 3 min at room temperature (15-25°C) * Resuspend pelleted bacterial cells in 250 μl Buffer P1 (resuspension buffer) and transfer to a microcentrifuge tube * Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue * Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. If using LyseBlue reagent, the solution will turn colorless * Centrifuge for 10 min at 13000rpm (17900\*g) in a table-top microcentrifuge * Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30-60s and discard the flow-through * Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30-60s and discard the flow-through * Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30-60s and discard the flow-through * Centrifuge for 1min to remove residual wash buffer * Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA add 50 μl Nuclease-Free water to the center of the QIAprep 2.0 spin column, let stand for 1min, then centrifuge for 1min * Store plasmids at -20°C Making competent cells▼ --- Day 1: Pre-culture * Inoculate 5 ml of LB medium in autoclaved glass tube with correct antibiotic with cells * Inucubate 13-15h at 37°, 120-150 rpm, 45° tilted * Autoclave a 250-500ml Erlenmeyer for the next day Day 2 * Re-inoculate E. coli into fresh LB medium by 1:100 dilution and cultivate at 37o C, 150 rpm (we did it with 120 rpm) for 2.5 - 3 h. Note: 200 mL of fresh culture is necessary to prepare 80-100 aliquots of competent cells (100 µL), leading to a 20-fold concentration. * Note: Since we want to make around 40 aliquots - starting a culture of 100 ml LB with 1 ml of the Pre-Culture should be enough (in a 250 ml Erlenmeyer flask) * 3. When OD reaches 0.2 - 0.3, cool down the cells to 4°C and harvest the cells at 4°C, 4200 rpm (max. for our centrifuge) for 15 min. Use 50 ml falcons for that. * Note: Set the centrifuge to 4°C and make a NOTE on the centrifuge indicating the necessary time for low temperature centrifugation. It takes about 30 min to cool down to 4°C. It is important to keep 4°C from this step and manipulate the cells gently - be careful to balance correctly the centrifuge From this point on keep the cells on ice at all times * Discard the supernatant and resuspend the cells with COLD 25 mL 0.1 M CaCl2 by pipetting, leave the cells with CaCl2 on ice for 5 min. * Note: CaCl2 is stored at 4°C. To further cool down CaCl2 it can be placed at -20°C 10min prior to use. * Centrifuge at 4200rpm for 10 min at 4°C * Discard the supernatant and gently resuspend the pellet in 4-5 mL of COLD 0.1 M CaCl2 with 25% glycerol * If you want to do transformations already today and use the cells fresh - it is recommended to resuspend those aliquots with CaCl2 0.1M without glycerol - therefore you would have to separate the part of the culture at step 4 or earlier. * Incubate the cells on ice for 10 minutes * Aliquot 100 µL of the competent cells to ice-cold 1.5 mL Eppendorf tubes (put them on ice previous to pipetting) and store them at -80o C. Note: (1) Some protocols require liquid nitrogen for this final cooling step, but it is NOT necessary. Directly transferring these competent cells from ice to -80o C freezer does not influence the transformation efficiency at all. (2) Fresh competent cells could be used on the same day of preparation by keeping them on ice. (3) Final concentration of glycerol could range from 15% to 25% Sequencing▼ --- Introduction The aim of the procedure is to determine the sequence of a plasmid or another DNA fragment Materials * 600-1500 ng Plasmid/DNA * 3 μl of the 20 µM primer * Sterile Water * 1.5 ml Eppendorf tube Procedure * Perform the plasmid isolation to isolate the plasmid that needs to be sequenced * In the 1.5 ml Eppendorf tube mix the plasmid (or other DNA fragment) with the corresponding primer * Add Sterile Water to the final volume of 15 μl * Label the tubes and send them to the Sequencing Microvolume Spectrophotometer▼ --- Introduction Microvolume Spectrophotometer is used to quantify the amount of DNA, RNA or Protein in a solution Materials * Pipette * Solution Procedure * Pipet a single drop (1 μl) of your DNA, RNA, or protein sample on the pedestal and pull down the arm * Let the spectrophotometer read the solution * Write down the concentration shown on the screen Colony PCR▼ --- Introduction Colony PCR to verify presence of PctD-LBD plasmid in BL21 DE3 plysS cells Materials * Primer 5' T7 promotor fw 10µM * Primer 3' T7 terminator reverse 10µM * Nuclease free water * LB plate with colonies to be checked for presence of plasmid * PCR tubes (200µl) and pen for labelling * Thermocylcer * Pipettes + Pipette Tips * Ice for cooling * When using GoTaq 2x Master Mix green: + GoTaq Green Master Mix, 2x * When using GoTaq polymerase (non-master mix) + OneTaq 5x buffer (green color) (promega) + OneTaq DNA polymerase (promega) + dNTP mix 10mM (2.5mM each dNTP) Procedure * Prepare PCR tubes according to the number of colonies to be screenedby labelling them and placing them on ice. Keep tubes on ice for the following steps. * PCR-Table **For using non master mix** | Reagent | 1x volume for 25 µl reaction (µl) | | --- | --- | | Go Taq buffer 5x | 5 | | dNTP mix 10mM | 0.5 | | FW primer 10µM | 1.25 | | RV primer 10µM | 1.25 | | Nuclease free water | 16.875 | | Go Taq DNA polymerase (5u/μl) | 0.125 | | | 25 | **For using 2x GoTaq Master Mix (green)** | Reagent | 1x volume for 25 µl reaction (µl) | 50x | | --- | --- | --- | | Go Taq Green Master Mix, 2x | 12.5 | 625 | | FW primer 10µM | 1.25 | 62.5 | | RV primer 10µM | 1.25 | 62.5 | | Nuclease free water | 10 | 500 | | | 25 | 1250 | * Under the clean bench: Mix all components of the PCR reaction apart from the colony one 1.5 ml tube. Add Polymerase last. After adding the polymerase, mix by vortexing shortly. On ice. * Transfer 25 µl of the master mix reaction to prepared PCR tubes. * Use pipette tip to pick a colony from the agar plate and streak them on the plate a little bit to dilute the amount of bacteria you transfer. Then place pipette tip into the correct PCR tube and swirl around. Discard pipette tip. Repeat for all colonies you want to screen. * Mix contents of PCR tubes by vortexing, then centrifuge with tabletob centrifuge. * Transfer PCR tubes to Thermocycler and run the following program: | A | Temperature | C | | --- | --- | --- | | Initial Denaturation | 95 °C | 5 min | | **30x cycle:** | | Denaturation | 95 °C | 30 s | | Primer annealing | depending on primer pair | 30 s | | Elongation | 72 °C | 1min /kb | | **cylce end** | | Final elongation | 72°C | 2x the normal elongation time (minimum 5 min) | | Cool to room temperature | 20 °C | infitnite | Gibson Assembly (commercial kit)▼ --- Introduction The goal is to assemble together different DNA fragments of our chimeric receptor with a backbone which harbours the reporter protein GFP Materials * Gibson MasterMix 2x * 0.08 pmol DNA (Vector and inserts) * Nuclease-free water * Thermocycler * Ice Procedure * Thaw GeneArt Gibson Assembly HiFi Master Mix on ice * Vortex GeneArt Gibson Assembly HiFi Master Mix immediately before use * In a microcentrifuge tube on ice, set up the GeneArt™ Gibson Assembly® cloning reaction as described below (1-3 Inserts Assembly): + Recommended DNA molar ratio vector:insert = 1:1 + Amount of each fragment 0.08 pmol vector 0.08 pmol each insert X µL + GeneArt™ Gibson Assembly® HF Master Mix 10 µL + Deionized water volume (10 – X) µL + Total volume 20 µL + Incubation time at 50°C 15 minutes * Mix the reactions by vortexing, spin down and incubate at 50°C for the recommended time. For Positive Control, use 15 minutes incubation time * After incubation, place the reaction mix on ice and immediately proceed to the transformation step Transformation (E.coli)▼ --- Introduction The aim is to introduce our plasmid of interest into bacteria cells Materials * Chemically competent E.coli * Plasmid DNA * Agar plates (with appropiate antibiotic) * S.O.C. medium * Incubator * Heating block * Ice Procedure * Thaw a tube of Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice. * Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex. * Place the mixture on ice for 30 minutes. Do not mix. * Heat shock at exactly 42°C for exactly 30 seconds. Do not mix. * Place on ice for 5 minutes. Do not mix. * Pipette 950 µl of room temperature SOC into the mixture. * Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate. Warm selection plates to 37°C. * Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC. * Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours Buffers and Media --- All concentrations are given as final concentrations unless stated otherwise. 10x PBS▼ --- 10x PBS▼ --- 10x PBS▼ --- Recipe taken from: <https://www.sigmaaldrich.com/GB/en/support/calculators-and-apps/1x-phosphate-buffered-saline> * 1330 mMNaCl * 27 mM KCl * 100 mM Na2HPO4 * 18 mM KH2PO4 * HCl for adjusting the pH * MilliQ H20 to 1 l 5x SDS loading buffer/dye▼ --- This is the recipe for a 5x SDS loading buffer. Use this 1+4 to create samples for SDS PAGE analysis. Remember to heat samples 95°C 5-10 min before loading. * 250 mM Tris/HCl pH 6.8 * 10 % SDS * 3 % glycerol * 5 % beta-mercaptoethanol * 0.02 % bromophenol blue 10x SDS running buffer▼ --- Recipe for 10x SDS. Recipe taken from: <https://www.aatbio.com/resources/buffer-preparations-and-recipes/sds-page-10x-sds-running-buffer> Concentrations given are final concentrations. Dilute with MilliQ water 1+9. * Tris 0.25 M * Glycine 1.9 M * Sodiumdodecyl sulfate (SDS) 1 % (w/w) * MilliQ H20 to 1 l 50x TAE buffer▼ --- * Tris 2 M * Glacial acetic acid 1 M * 0.05M EDTA, pH 8.0 * MilliQ water to 1L HisTrap IMAC binding buffer▼ --- * 30 mM Tris/HCl * 300 mM NaCl * 10 mM imidazole * 5% [vol/vol] glycerol * adjust pH to 8.0 with HCl * -> filter through 0.45µm filter with peristaltic pump HisTrapIMAC elution buffer▼ --- * 30 mM Tris/HCl fo * 300 mM NaCl * 500 mM imidazole * 5% [vol/vol] glycerol * adjust pH to 8.0 with HCl * -> filter through 0.45µm filter with peristaltic pump HisTrapIMAC resuspension buffer▼ --- * 30 mM Tris/HCl * 300 mM NaCl * 5% [vol/vol] glycerol * MilliQ water ITC buffer▼ --- Buffer for ITC measurement of PctD-LBD protein according to <https://journals.asm.org/doi/10.1128/mbio.03458-21> * 30 mM Tris, * 5 mM PIPES [piperazine-N,N9-bis(2-ethanesulfonic acid)] disodium salt monohydrate * 5 mM MES (morpholineethanesulfonic acid) hydrate * 150 mM NaCl * 10% (vol/vol) glycerol * pH 7.5 LB/LB-agar (Miller) medium (1L)▼ --- * 10g Tryptone * 10g NaCl * 5g Yeast Extract * 15g agar (only for LB-agar medium for plates) * 1L Milli-Q or distilled water M9 minimal medium (100 mL)▼ --- * M9 salts (5X) - 20 mL * Glucose (20%; Sigma-Aldrich)a - 2 mL * MgSO4 (1 M; Fisher Scientific)b - 200 μL * CaCl2 (1 M; Fisher Scientific)b - 10 μL * H2O - 78 mL (final volume 100 ml) * a: Filter-sterilize and store at 4°C. * b: Autoclave and store at room temperature. M9 salts 5x (1L)▼ --- * Na2HPO4•7H2O (M=268,07 g·mol−1), 64 g + -> we have water-free Na2HPO4 (M= 141,96 g·mol−1 ) + -> use 33.89g instead * KH2PO4, 15 g * NaCl, 2.5 g * NH4Cl, 5.0 g * deionized H2O, to 1 liter * Divide the salt solution into 200-ml aliquots and sterilize by autoclaving for 15 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle. LBD-PctD experiments Expression of PctD-LBD▼ --- Preparation * Isolate plasmid containing PctD-LBD acording to plasmid isolation protocol * Transform BL21 DE3 PLysS cells with isolated plasmid acording to transformation protocol * Plate transformed cells on LB agar plates substituted with chloramphenicol and kanamycine Day 1: Pre-Culture * Inoculate 100 ml LB medium (medium and erlenmeyer flask autoclaved!) substituted with kanamycine (25 µg/mL) and chloramphenicol (17 µg/ml) with 1 single culture of transformed cells * incubate overnight at 37°C while shaking Day 2: Main Culture * Measure OD600 of pre-culture * Inoculate 500 ml LB medium (medium and erlenmeyer flask autoclaved!) substituted with kanamycine (25 µg/mL) and chloramphenicol (17 µg/ml) with main culture * Incubate main culture at 37°C while shaking until OD600 = 0.5 * Take sample for SDS PAGE * induce expression by addition of 1 mM IPTG * Grow for 5 hours at 37°C while shaking * Take sample for SDS PAGE * Measure OD600 * harvest cells: + transfer cell suspension to centrifugation containers (250 ml fit in each) + centrifuge at 20,000 g for 20 min at 4°C * Freeze cells Cell lysis▼ --- Note: keep proteins/cell suspension on ice at all times * Thaw cell pellet on ice * Resupend cell pellet + add 20 ml binding buffer + dissolve 1 tablet of protease inhibitor + Add DNAse I to final concentration of 500 ng/ml or 0.2 U/ml (2 U total) + resuspend cells + transferred protein suspension to 50 ml falcon tube * lyse cells by sonication: + keep cells on ice while sonication + performe sonication with "4 x 4 x 4 x4" settings: - 4 cycles - 4 minutes on, 4 min pause between cylces - 40 % duty cycle - power output: 4 * Centrifuge at 20000 x g for 20 min at 4 degrees. * filter supernatent through 0.45 micrometer filter using a syringe * Take sample for SDS PAGE Protein Purification▼ --- Used for purification: Äkta FPLC column: 5ml HisTrap, flow rate: 2 ml/min Buffer A: Binding buffer Buffer B: Elution buffer * wash with 15 column volumes buffer A * Take sample for SDS PAGE * Apply gradient from 0% buffer B to 100% buffer B over 25 column volumes-> fractionated during elution in 1 ml fractions * cleaning: + wash with buffer A + wash with water + wash with 20 % ethanol * Confirm purification success by SDS PAGE Dialysis▼ --- * cut piece of semipermeable membrane hose (Dialysemembran Spectra/Por , MWCO 3500 Da) * soak membrane hose in water for 10 min * use a clip to close hose from one end * add protein solution to the hose from the open end with a pipette tip * close second end of the hose with another clip * fill ITC buffer into 1l glass beaker * add stirring flea * attach clips to a styropor floater ring and place hose into beaker * place beaker onto a magnetic stirrer in 4 °C room * stirr over night (at least 16h) with ca. 250 rpm ITC▼ --- Materials * Malvern MicroCal PEAQ-ITC * 300µl PctD -LBD in ITC buffer (c = 13.5 \* Kd) * 40µl choline in ITC buffer (c = 14\* protein concentration) * ITC buffer for washing Procedure * Prepare 300 µl protein solution (c = 13.5 \* Kd) * Prepare 40 µl ligand solution (c = 14\* protein concentration) * Degased all solutions utilizing a vacuum pump. * Wash the chamber of the ITC instrument with buffer 3x * Rinse the injection needle with ITC buffer. * Fill the chamber with protein solution. * Fill injection needle with ligand. * Set the following instrument settings: | Temperature (C°) | 25 | | --- | --- | | Reference power (µcal/s) | 10 | | Feedback | High | | Stir speed (rpm) | 750 | | Initial Delay (s) | 60 | | Injection spacing (s) | 150 | | Injection duration (s) | 4 | Chimeric receptor development --- DNA fragments amplification (PCRs)▼ --- Pctd | DNA polymerase | Component | Volume [µL] | | --- | --- | --- | | Pfu | Pfu buffer 10x | 5 | | Pfu | 0.5 | | Q5 GC enhancer 5x | 10 | | dNTPs Mix | 1 | | Primer IGT\_002 | 2.5 | | Primer IGT\_006 | 2.5 | | Nuclease-free H2O | 27.5 | | DNA template | 1 | | Total | 50 | | Q5 | Q5 High-fidelity MasterMix 2x | 12.5 | | Q5 GC enhancer 5x | 5 | | Primer IGT\_002 | 1 | | Primer IGT\_006 | 1 | | Nuclease-free H2O | 4.5 | | DNA template | 1 | | Total | 25 | Program (thermocycler): | Step | Temperature [°C] | Time   [sec] Pfu/Q5 | Cycle | | --- | --- | --- | --- | | Initial   denaturation | 95 | 120 | 1 | | Denaturation | 95 | 30 | 30 | | Annealing | 58 | 30 | 30 | | Elongation | 72 | 180/90 | 30 | | Final   elongation | 72 | 300/120 | 1 | | Storage | 20 | ∞ | | Pctd-HAMP | DNA polymerase | Component | Volume [µL] | | --- | --- | --- | | Pfu | Pfu buffer 10x | 5 | | Pfu | 0.5 | | Q5 GC enhancer 5x | 10 | | dNTPs Mix | 1 | | Primer IGT\_006 | 2.5 | | Primer IGT\_008 | 2.5 | | Nuclease-free H2O | 27.5 | | DNA template (linearized pkg116) | 1 | | Total | 50 | | Q5 | Q5 High-fidelity MasterMix 2x | 12.5 | | Q5 GC enhancer 5x | 5 | | Primer IGT\_006 | 1 | | Primer IGT\_008 | 1 | | Nuclease-free H2O | 4.5 | | DNA template | 1 | | Total | 25 | Program (thermocycler): | Step | Temperature [°C] | Time [sec] Pfu/Q5 | Cycle | | --- | --- | --- | --- | | Initial denaturation | 95 | 120 | 1 | | Denaturation | 95 | 30 | 30 | | Annealing | 58 | 30 | 30 | | Elongation | 72 | 210/90 | 30 | | Final elongation | 72 | 300/120 | 1 | | Storage | 20 | ∞ | | Envz | DNA polymerase | Component | Volume [µL] | | --- | --- | --- | | Pfu | Pfu buffer 10x | 5 | | Pfu | 0.5 | | dNTPs Mix | 1 | | Primer IGT\_003 | 2.5 | | Primer IGT\_004 | 2.5 | | Nuclease-free H2O | 37.5 | | DNA template | 1 | | Total | 50 | | Q5 | Q5 High-fidelity MasterMix 2x | 12.5 | | Primer IGT\_003 | 1 | | Primer IGT\_004 | 1 | | Nuclease-free H2O | 9.5 | | DNA template | 1 | | Total | 25 | Program (thermocycler): | Step | Temperature [°C] | Time [sec] Pfu/Q5 | Cycle | | --- | --- | --- | --- | | Initial denaturation | 95 | 120 | 1 | | Denaturation | 95 | 30 | 30 | | Annealing | 58 | 30 | 30 | | Elongation | 72 | 120/30 | 30 | | Final elongation | 72 | 150/45 | 1 | | Storage | 20 | ∞ | | EnvZ-Hamp | DNA polymerase | Component | Volume [µL] | | --- | --- | --- | | Pfu | Pfu buffer 10x | 5 | | Pfu | 0.5 | | dNTPs Mix | 1 | | Primer IGT\_003 | 2.5 | | Primer IGT\_004 | 2.5 | | Nuclease-free H2O | 37.5 | | DNA template | 1 | | Total | 50 | | Q5 | Q5 High-fidelity MasterMix 2x | 12.5 | | Primer IGT\_003 | 1 | | Primer IGT\_004 | 1 | | Nuclease-free H2O | 9.5 | | DNA template | 1 | | Total | 25 | Program (thermocycler): | Step | Temperature [°C] | Time [sec] Pfu/Q5 | Cycle | | --- | --- | --- | --- | | Initial denaturation | 95 | 120 | 1 | | Denaturation | 95 | 30 | 30 | | Annealing | 58 | 30 | 30 | | Elongation | 72 | 120/30 | 30 | | Final elongation | 72 | 150/45 | 1 | | Storage | 20 | ∞ | | Pkg116 | DNA polymerase | Component | Volume [µL] | | --- | --- | --- | | Pfu | Pfu buffer 10x | 5 | | Pfu | 0.5 | | dNTPs Mix | 1 | | Primer IGT\_005 | 2.5 | | Primer IGT\_007 | 2.5 | | Nuclease-free H2O | 37.5 | | DNA template (linearized pkg116) | 1 | | Total | 50 | | Q5 | Q5 High-fidelity MasterMix 2x | 12.5 | | Primer IGT\_005 | 1 | | Primer IGT\_007 | 1 | | Nuclease-free H2O | 9.5 | | DNA template (linearized pkg116) | 1 | | Total | 25 | Program (thermocycler): | Step | Temperature [°C] | Time [sec] Pfu/Q5 | Cycle | | --- | --- | --- | --- | | Initial denaturation | 95 | 120 | 1 | | Denaturation | 95 | 30 | 35 | | Annealing | 58 | 30 | 35 | | Elongation | 72 | 540/180 | 35 | | Final elongation | 72 | 600/240 | 1 | | Storage | 20 | ∞ | | Plasmid assembly (Gibson assembly)▼ --- Following recommended protocol on the manual: Mix an equimolar DNA amount of inserts and vector (0.08pmol each) | Component | Volume [µL] | | --- | --- | | -    Gibson MasterMix 2x | 10 | | -    DNA fragments (insert&backbone) | X | | -    Nuclease-free H2O | (10-X)\* | \*Fill up until final volume of 20µL * Mix all components (vortexing) * Incubation 15min at 50°C * Place the reaction mix on ice and proceed to the transformation Transformation into E.coli strains (DH5a, VS1007)▼ --- * Thaw a tube of Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice. * Add 1µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex. * Place the mixture on ice for 30 minutes. Do not mix. * Heat shock at exactly 42°C for 30-40 seconds. Do not mix. * Place on ice for 5 minutes. Do not mix. * Pipette 950 µl of room temperature SOC into the mixture. * Place at 37°C for at least 60 minutes. Shake vigorously (250 rpm) or rotate. Warm selection plates to 37°C. * Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions and concentration in SOC. * Spread 100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours Testing of Biosensor --- Bacterial growth curves ▼ --- Day 1 * Inoculate LB medium (supplemented with strain-specific antibiotics) with E.coli * Let grow overnight at 37°C while shaking at 200 rpm Day 2 * Inoculate 12 ml LB medium (supplemented with strain-specific antibiotics) with 250µl overnight culture * Transfer 1ml of culture into cuvette and measure OD600 with Photospectrometer (t0) * incubate remaining culture at 37°C while shaking at 200 rpm * Repeat steps 4 and 5 every hour for at least 6h Fluorescence microscopy▼ --- Day 1 * Inoculate M9 medium (supplemented with strain-specific antibiotics) with E.coli * Let grow overnight at 37°C while shaking at 200 rpm Day 2 * Transfer 1ml of culture into cuvette and measure OD600 with Photospectrometer * IF OD600 < 0.5: spin down culture and resuspend cell pellet in less M9 medium * IF OD600 >= 0.5: add Salicylic acid (effective concentration: 600µM) to induce protein expression * Incubate at 37 °C while shaking at 200 rpm At least 1h before fluorescence microscopy: * Transfer 50 µl of each culture into well of 96-well-plate * Add ligand to an effective concentration of 200m Fluorescence microscopy: * Transfer 1 drop of each culture on to agarose slide * Cover samples with coverslips * Image slides with fluorescence microscope Quantification of Fluorescence in Plate Reader▼ --- Outline: * Start 10ml over night cultures of cells in LB medium in glass tubes. * Measure OD600 the next day with spectrohotometer * Dilute cultures to an OD600 of 0.1 in polypropylene 15 ml falcons in a final volume of 6 ml in M9 medium. * Grow until OD600 reaches 0.5. Supplement M9 medium with acetylsalicylic acid and acetycholine/choline. * Measure green fluorescence and absorbence at 600nm with plate reader * Incubate over the course of 16h. Take samples to measure absorbance at 600nm and green fluorescence at 2h, 4h, 6h, 8h, 16h Day 1 * start 10 ml overnight cultures for the strains to be tested in glass tubes with M9 medium supplemented with antibiotics * incubate at 37°C, 200 rpm overnight Day 2 * place glass tubes on ice to stop growth * remove 1ml medium to measure OD600 with spectrophotometer * measure OD600, then dilute cultures into M9 minimal medium to an OD600 of 0.2 in 15ml polypropylene tubes * mix by swirling, remove 1 ml medium for control measurement of OD600 * incubate cultures in M9 minimal medium at 37°C, 200 rpm until OD600 reaches 0.5 * place tubes on ice to stop growth * Measure OD600 again; then add Acetylsalicylic acid (2 mM stock, 4μM final concentration) to all cultures and choline or acetlycholine to designated samples * add 700 μl choline or acetylcholine of 10x stock solutions (3 different stock solutions for each acetylcholine and choline at 100μM, 1mM, 10mM) * add700μl MilliQ water, sterile filtered to samples with no choline or acetylcholine added to have the same volume in all cultures * take samples for measurement with plate reader: * remove 0.6 ml of each culture and transferre it to 1.5ml tubes on ice * continued incubation of cultures + meanwhile: pipet 200 μl of samples for plate reader measurement of absorbance at 600nm and green fluorescence + Incubate cultures at 37°C, 200 rpm for 16h. Take samples to measure absorbance at 600nm and green fluorescence at 2h, 4h, 6h, 8h, 16h ![Plate Layout](https://static.igem.wiki/teams/4208/wiki/experiments/plate-layout.png) Figure 1: Plate Layout Drylab --- Binding Site Prediction with SSnet▼ --- Install SSnet from <https://github.com/ekraka/SSnet> Preparation of Protein: * Download Pctd as .pdb File from NCBI (7PRR) * Remove Acetylcholine from it with PyMol Preparation of Ligands: Use SMILE format * Anatoxin A = CC(=O)C1=CCCC2CCC1N2 * Acetylcholine = CC(=O)OCC[N+](C)(C)C * Betaine = C[N+](C)(C)CC(=O)[O-] * Choline = C[N+](C)(C)CCO Executing via command line: SSnet\_windows.exe -t 7PRRwithoutACh.pdb -l CC(=O)C1=CCCC2CCC1N2 (for Anatoxin A, and p-value) SSnet\_windows.exe -t 7PRRwithoutACh.pdb -l CC(=O)C1=CCCC2CCC1N2 -m grad (for heatmap of binding site) The heatmap is then safed in a pdb file an can be visualized in PyMol with the command spectrum b, rainbow, minimum = 0.0, maximum = 100.0 References: * Verma, N.; Qu, X.; Trozzi, F.; Elsaied, M.; Karki, N.; Tao, Y.; Zoltowski, B.; Larson, E.C.; Kraka, E. SSnet: A Deep Learning Approach for Protein-Ligand Interaction Prediction. Int. J. Mol. Sci. 2021, 22, 1392. <https://doi.org/10.3390/ijms22031392> SeeSar affinity prediction▼ --- Install SeeSar from <https://www.biosolveit.de/products/seesar/> Preparation of Protein: * Download Ptcd as pdb File from NCBI (7PRR) or select straight from inside the app Preparation of Ligands: * Download Anatoxin A, Acetylcholine, Betaine and Choline as SDF file from PubChem Detecting Binding Site: * Select Protein to work with in Protein Mode, than change to Binding Site Mode and detect all binding sites * Select binding Site where Acetylcholine is inside Molecular Docking: * Change to Docking Mode: * Select or upload Ligand which should be docked inside pocket * Change sphere size and number of molecule positions * Run docking * You can sort the results by best affinity and download them as pdb file to further inspection Molecular Dynamic Simulation with GROMACS▼ --- Install GROMACS as adivced by their installation guide (<https://manual.gromacs.org/current/install-guide/index.html>) Either use the Linux subsystem for Windows or install it straight on Linux (in our case Ubuntu 18, but there are some packages missing on it). Follow the Tutorial from bioinformatics review (<https://bioinformaticsreview.com/20191210/tutorial-molecular-dynamics-md-simulation-using-gromacs/>). To view the Results as Video: * Install VMD for Ubuntu. * Open VMD and load md\_0\_1.gro into it. * Then select a trajectory file, ending with .trr or xtc. * In Graphics and representation replace all by protein so you can only see the protein. Then press play.

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### Chimeric receptor development

---

#### Introduction

In our initial experiments we aimed to assemble our chimeric receptor which will be able to sense and bind to the target Anatoxin-a. In order to achieve this, as first step we amplified by PCR using self-designed primers the DNA sequence of the chemoreceptor Pctd sensing domain, the EnvZ signaling domain of the EnvZ/OmpR two-component system, and the vector backbone which harbors the reporter protein GFP. Afterwards, all amplified DNA fragments were assembled together via the Gibson assembly technique and the new plasmid was transformed into our final *E. coli* strain. Lastly, the *E. coli* biosensor was tested in the presence of several compounds to assess its sensitivity and reliability.

![Structure of anatoxin-a](https://static.igem.wiki/teams/4208/wiki/chimeric-receptor-development/schaubild.png)

Figure 1: Schematic overview of chimeric receptor development via Gibson assembly technique.

#### Results

**DNA fragments amplification (PCRs)**

Fig.2 shows the agarose gel of the different amplified DNA fragments by PCR, which were assembled afterwards. All fragments were successfully amplified as the bands seem to have the expected size.

In lanes 2 and 3 are found two different fragments of the EnvZ response regulator. The larger one in lane 2, with a size of 842bp, contains the HAMP domain which has been described as essential in the signaling process of the two-component system. The other lacks the HAMP domain and has a smaller size of 708bp.

The HAMP domain connects the periplasmic ligand-binding domain (LBD) of the chemoreceptor Pctd to the cytoplasmic signaling domain, thus it plays an important role in the transmission of sensory signals. Indeed, the packing stability of the HAMP domain has been described to be highly critical to signaling and any displacement of its structural integrity could lead to a non-functional sensory protein. Additionally, extensive mutational analysis has shown that a single point mutation in the HAMP domain can result in the modification of the receptor ligand specificity as well as the constitutive state, therefore the integrity of HAMP becomes crucial.

In our approach, since we aim to generate a hybrid receptor from Pctd and EnvZ, and both of them have their own HAMP domain, we have generated different DNA fragments which might contain or not the HAMP domain that will be fusioned together and assessed to obtain the chimeric receptor with the highest output.

Lanes 4 and 5 show the sensor domain of the chemoreceptor Pctd. Like EnvZ fragments, the pctd gene was amplified twice, with and without the HAMP domain, with sizes of 1365bp and 1195bp, respectively.

Lastly, the backbone pkg116 is shown in lane 6 with an expected size of 4228 bp. It will be used as our reporter plasmid in which the GFP fluorescence will serve as signal to indicate the presence of the toxin. The different inserts of the chimeric receptor will be assembled with this vector.

![PCR agarose gel](https://static.igem.wiki/teams/4208/wiki/chimeric-receptor-development/gel-3.png)

Figure 2: Agarose gel electrophoresis of the different DNA fragments amplified by PCR. All PCRs were carried out succesfully as all bands have the expected length. Lanes 2 and 3 show the EnvZ gene without and with the HAMP region, respectively. Lanes 4 and 5 show the pctd gene without and with the HAMP region, respectively. Lane 6 show the vector pkg116 which will be assembled with the different inserts.

 **Plasmid assembly (Gibson + sequencing & gel electrophoresis)**

The Gibson assembly method was performed to generate our construct which harbors the chimeric receptor. Following this purpose, 2 different receptors were assembled and transformed directly into chemically competent DH5a cells: PHE (pctd-HAMP + EnvZ + pkg116), in which the HAMP domain comes from the pctd gene, and PEH (pctd + EnvZ-HAMP + pkg116), where the HAMP domain is natively from the EnvZ gene.

Both receptors will be assessed to figure out which has the higher output and therefore we could determine which HAMP domain should be used, either from the receptor or the response regulator, in future attempts to generate new hybrid receptors.

**Transformation into *E. coli* strains (+ colony PCR)**

###### DH5a cells

The first bacterial strain used for transformation of our assembled plasmid was E-coli DH5a cells. This strain was used due to their high transformation efficacy, which allowed us to obtain a great number of transformed colonies to perform a colony PCR.

Figures 3 and 4 show the electrophoresis gel of a colony PCR of the DH5a cells transformed with the assembled PHE and PEH, respectively. Interestingly, the assembly of PEH (Fig 4) seemed to be more efficient than PHE (Fig. 3) as the proportion of DNA bands – each coming from one different DH5a colony (Fig.5) – with the expected length of 2.2kb is higher.

Furthermore, the PHE and PEH plasmids were also isolated through by miniprep and sent for sequencing to check and verify the correct orientation of the inserts. The results (not shown) confirmed that the inserts were assembled in the correct orientation.

![PHE assembly agarose gel](https://static.igem.wiki/teams/4208/wiki/chimeric-receptor-development/gel1.png)

Figure 3: Colony PCR of PHE plasmid in transformed DH5a E. coli cells. Lanes 6-8 and 10 show the PHE region as the bands seem to have the expected length of 2.2 kb. The negative control is found in lane 3.

![PEH assembly agarose gel](https://static.igem.wiki/teams/4208/wiki/chimeric-receptor-development/gel-2.png)

Figure 4: Colony PCR of PEH plasmid in transformed DH5a E. coli cells. Lanes 4-12 show the PEH region as the bands seem to have the expected length of 2.2 kb.

![DH5 alpha transformation with hybrid receptor](https://static.igem.wiki/teams/4208/wiki/chimeric-receptor-development/colonies.png)

Figure 5: Transformed DH5a cell colonies grown on LB-agar + chloramphenicol plate. Colonies marked in red were used for colony PCR. (A) DH5a cell colonies transformed with PHE plasmid. (B) DH5a cell colonies transformed with PEH plasmid

###### VS1007

Once it was ensured that the plasmid was assembled and transformed successfully into DH5a cells, we proceeded to transform both plasmids in our final *E. coli* strain VS1007 which carries the reporter plasmid harbouring GFP. Like in DH5a cells, we also performed a colony PCR as well as sequencing to verify the integrity of our plasmids.

In Figure 6 it is shown the gel electrophoresis of colony PCR and it can be seen in lanes 6 and 7 the positive colonies carrying the PEH plasmid and PHE plasmid, respectively.

![VS1007 transformed with hybrid receptors](https://static.igem.wiki/teams/4208/wiki/chimeric-receptor-development/gel-4.png)

Figure 6: Colony PCR of PEH and PHE plasmids in transformed VS1007 E. coli cells. Lanes 6 and 7 show the PEH and PHE regions, respectively, as the bands seem to have the expected length of 2.2 kb.

#### Conclusion & future prospects

We successfully developed our biosensor which is based on the *E. coli* strain VS1007, that carries the reporter plasmid harboring GFP, by first amplifying and assembling the DNA sequences of our chimeric receptor via Gibson assembly, followed by transformation of the receptor into this bacterial strain.

Owing to the essential role of the HAMP domain in the signaling pathway triggered upon ligand binding, as well as the fact both proteins of our hybrid receptor – Pctd and EnvZ – have their own HAMP domain, we generated two versions of our chimeric receptor, PEH and PHE, that will be assessed in following experiments to obtain the one with higher output.

Strikingly, sequencing results revealed the presence of a P203S mutation in the chimeric receptor, and the same mutation was also found in the original Pctd template DNA. This amino acid is close to the binding pocket of Pctd, thus it may affect negatively the affinity of the receptor to its ligand.

Under this circumstance, we proceeded to assess the consequences of this unexpected mutation in the specificity and reliability of our biosensor. Furthermore, if the results of our experiments testing the biosensor are not good, we have planned to remove the mutation by using self-designed primers to PCR out this mutation near the binding site from the beginning, thereby obtaining a new version of our biosensor.

### Drylab results

---

In order to produce a chimeric receptor which Anatoxin-a can bind to, we tried to predict the binding affinity of Antoxin-a to PctD in silicio. As first step we predicted the binding site correlating with Anatoxin-a. This was done with SSNet [(1)](#1).

#### SSnet

| Molecule | p-Value |
| --- | --- |
| Acetylcholine 0.02057565189898014 | |
| Choline  0.054201576858758926 | |
| Betaine   0.04926067963242531 | |
| Anatoxin-a  0.15658815205097198 | |

It has previously been shown that ACh, Choline and Betaine all bind the same binding sitei. In our modelling with SSnet Anatoxin-a seems has a lower possibility to bind there compared to the natural ligands(highest p-value). Still SSnet was predicting the binding site of Anatoxin-a as the same as the one from ACh.

![SSnet results for Anatoxin-a](https://static.igem.wiki/teams/4208/wiki/project/results/drylab-results/ssnetanatoxina.png)

Figure 7: Binding sites predicted with SSnet for Anatoxin-a.

In Figure 7 you can see the positions of Pctd where SSnet predicted some binding possibility. In red are the positions where Anatoxin-a is most likely to bind, those are the same positions as for ACh and the other tested molecules. Because the protein is molecular dynamic, the two bidning sites at each of the same chains have not the same quality for binding. That leads to the next step: Predict the binding of Anatoxin-a.

In the second step, we then predicted the binding affinity of Anatoxin-a and other ligands using SeeSar [(2)](#2)

.

#### SeeSar

In Figure 8 is the binding prediction with the highest affinity predicted by SeeSar for Acetylcholine. As you can see, Acetylcholine is sitting deep inside of the binding site (blue-light molecule). Figure 9 is the highest affinity prediction for Anatoxin-a around the binding site, there were other predictions at the intacellular site of PctD.
Compared to Acetycholine, Betaine and Choline, Anatoxin-a doesn’t really bind inside of the binding site, sitting way outside of it. It seems that Anatoxin-a is too big to enter it.

![SeeSar binding site prediction for Acetylcholine](https://static.igem.wiki/teams/4208/wiki/project/results/drylab-results/seesarach.png )

Figure 8: Binding site predicted by SeeSar for Acetylcholine.

![SeeSar binding site prediction for Anatoxin-a](https://static.igem.wiki/teams/4208/wiki/project/results/drylab-results/seesaranatoxina.png)

Figure 9: Binding site predicted by SeeSar for Anatoxin-a.

Betaine and Choline are binding at the same location as Acetylcholine. These are the results of SeeSar with the highest binding affinity.

#### GROMACS

Since the results from both tools suggested binding of Anatoxin-a to PctD is considerably weaker compared to the natural ligands, we questioned wether predictions of Anatoxin-a binding would be more promising when accounting for dynamic conformations of the PctD receptor. We collaborated with iGEM Team  [iGEM Team Patras](https://2022.igem.wiki/patras/)  to carry out molecular dynamics simulation using Gromacs [(3)](#3).

In the Video of the Molecular Dynamic Simulation Anatoxin-a is flipping between the intracellular site (Figure 10)that was also predicted by SeeSar and outside of the real binding site (Figure 11), also like SeeSar predicted.

Even with Molecular Dynamic Simulation, there seems to not be enough space inside of the binding site for Anatoxin-a, even GROMACS has difficulties finding the correct binding sites. Next step would be to try to define the binding site of the protein before the simulation. But when you focus on those binding sites, you can see that they are not really flexible, what makes the chance for the big Anatoxin-a molecule to enter it way smaller.

![GROMACS result 1 for Anatoxin-a](https://static.igem.wiki/teams/4208/wiki/project/results/drylab-results/gromacsone.png)

Figure 10: Molecular Dynamic Simulation. Anatoxin-a can be found at the intracellular site of the receptor.

![GROMACS result 2 for Anatoxin-a](https://static.igem.wiki/teams/4208/wiki/project/results/drylab-results/gromacs2.png)

Figure 11: Molecular Dynamic Simulation. Anatoxin-a can be found near the binding site, but outside of the binding pocket.

#### Conclusion

After the in silicio analyisis, it is not likely that Anatoxin-a binds to PctD. Initially we wanted to do some mutational analysis, to improve binding of Anatoxin-a to PctD but Prof Thiel explained us that prediction of the effect of mutations on protein structures *in silicio* is currently too unreliable yet. This is because accurate protein structure prediction algorithms mostly rely on finding similar second structures in homologous proteins. Since the amount of known structures of homologous proteins is considerably lower when you introduce point mutations, the algorithms cannot reliable predict the effect of the point mutation.

Therefore, screening of mutated versions of PctD needs to be carried out in wetlab.

### PctD-LBD experiments

---

#### Introduction

PctD is a scarcely characterized chemoreceptor, found in Pseudomonas aeruginosa. It has been found that it binds to choline and acetylcholine.
[(4)](#4) Anatoxin-a mimics acetylcholine and binds to the nicotinic Acetylcholine receptors
[(5)](#5). To our knowledge, Anatoxin-a has not yet been shown to bind PctD. Since we wanted to use it for our biosensor, we wanted to test the binding of Anatoxin-a to PctD.

Therefore, we planned to express the PctD ligand binding domain (PctD-LBD) in BL21(DE3)pLys E.coli, purify it and use isothermal titration calorimetry (ITC) to test the binding of Anatoxin-a to the protein. This is important to establish, because the functionality of our biosensor is dependent on detection of Anatoxin-a by the PctD ligand binding domain.

We used a plasmid, that was kindly gifted to us by the lab of Victor Sourjik, that contained the His-tagged PctD-LBD to transform BL21(DE3)pLys E.coli cells and overexpress the protein [(4)](#4).

To purify the protein we used a HisTrap column and the Äkta-FPLC system. We used SDS gel electrophoresis to analyze the expression and purification.

Next, we performed ITC with the purified PctD-LBD and choline as a ligand. This was meant to be a positive control for our ITC experiment with Anatoxin-a as a ligand. Unfortunately, due to delivery issues, Anatoxin-a was not available for us to perform experiments with.

#### Results

 **Protein purification**

Purification with HisTrap column yielded two peaks at approximately 70 mM imidazol and 90 to 110 mM imidazol. The peaks are indicated with black arrows in Figure 12. To confirm that one of the peaks contains the purified PctD-LBD, samples of selected fractions of both peaks were analyzed with an SDS-PAGE alongside samples of the expression process (Fig.13).

![Chromatogramm](https://static.igem.wiki/teams/4208/wiki/project/results/pctd-purification-chromatogram.png )

Figure 12: Chromatogram of purification of PctD-LBD with HisTrap column. Green: gradient of elution buffer (0-100% buffer B), blue: UV signal, red: elution fractions (1 ml). The UV signal shows 2 peaks that are indicated by black arrows.

![SDS PAGE of expression](https://static.igem.wiki/teams/4208/wiki/project/results/pctd-purification-sdsgel.png )

Figure 13: Image of 12% SDS gel, stained with Coomassie blue solution. It contains samples from expression and purification of PctD-LBD (MW = 39.5 kDA). Legend: M = protein marker, L = total cell lysate, S = soluble fraction, P = pellet/insoluble fraction, E27 – E57: elution fractions. The protein sizes of the marker are indicated in kDA on the left. Lanes L, P, E54 and E57 contain bands of the expected size of 39.5 kDA (black boxes), indicating that the expression was successful and Peak 2 of the purification contains PctD-LBD.

The PctD ligand binding domain with a His-tag has a molecular weight of 39,5 kDa. Bands of the correct size can be seen in the SDS gel in the lanes “L” and “P”, that contain the whole cell lysate and the pellet of the unsoluble fraction, respectively. Bands of the same size can be seen in the lanes of the elution fractions 54 and 57. As expected, all of those bands are thicker than the rest of the visible bands, because the PctD ligand binding domain was overexpressed in our cells. This confirms, that Peak 2 contains the PctD-LBD protein.

The lanes of fractions E54 and E57 show further bands, suggesting that our target protein still contains some contaminations. But the bands are thin and faint, so we considered the fractions pure enough to work with the proteins further.

The fractions of Peak 2 were pooled and a dialysis against ITC buffer was performed. The protein solution was then pooled with the protein we obtained from another successful protein purification (following the same protocol).

The protein concentration was measured with a microvolume spectrophotometer and yielded a concentration of 2.5 mg/ml.

 **Isothermal titration calorimetrie**

For the ITC measurement, we prepared our protein (PctD-LBD) in a concentration of 35µM and choline in a concentration of 490µM in ITC buffer. These concentrations were chosen so that the protein concentration of the protein is between 10-100\*Kd (Kd = dissociation constant of ligand-protein interaction), and the final concentration of ligand is 2-times the protein concentration. The dissociation constant (Kd = 2.6 µM) was known from literature [(4)](#4).

During the ITC measurement, 40 µl of ligand was injected in 2µl doses in intervals of 2,5 min. Temperature changes for each injection were recorded.

If binding of ligand occurs, temperature spikes for each injection are expected, with decreasing peak area over time. This is because the binding sites of the receptors get saturated over time.

Our raw data is shown in Figure 14. The rate of heat release DP in µcal/s is plotte against the time. After integration the peak area is obtained. This corresponds to the enthalpy change dH. When plotting the enthalpy change against the molar ratio, a saturation curve is expected. This is visualized in Figure 15.

You can see that there is no continuous decrease in peak area. The first two peaks have a higher peak area and therefore a higher enthalpy change, the rest of the peaks are of similar, smaller size. Therefore, potting the enthalpy against the molar ratio (Fig. 15) does not show an optimal saturation curve, because the slope consists of only 1 data point.

The data we got could be explained by the fact that the ligand binding is not sufficiently exothermic, so that the signal intensity is too low to be observed. But it has already been shown by others that binding of Choline to PctD-LBD can be detected and quantified by ITC.

Another possible explanation for this is that our effective protein concentration (concentration of functional protein that can bind the ligand) was much lower than the total protein concentration in the solution. This might have resulted in the ligand binding site being saturated very fast, therefore explaining the raw data we got.

Additionally, the analysis software corresponding to the ITC system, was not able to fit a curve to our data and output information about the dissociation constant and other variables, because there was too little heat overall.

Instead, we decided to fit a curve to the data by hand (Fig. 16). We set the dissociation constant from the literature [(4)](#4) and the concentrations of our ligand and protein as constants, allowing us to vary the number of binding sites of our protein (N(sites)). The detailed fitting parameters can also be found in Figure 16. The curve that fitted our data had a value of N(sites) = 0.01 per protein. This corresponds to only 1% of present protein having a binding site for Choline, supporting our hypothesis that only part of our protein is functional.

![Raw data ITC experiment](https://static.igem.wiki/teams/4208/wiki/project/results/220916-pctd-lbd-choline-analysis-screenshot-dp.png )

Figure 14: Raw data of isothermal titration calorimetry. DP (µcal/s) is plotted against the time (s). Each peak corresponds to 1 injection of 2µl of choline.

![Enthalpie change per injection](https://static.igem.wiki/teams/4208/wiki/project/results/220916-pctd-lbd-choline-analysis-screenshot-delta-h.png)

Figure 15: Enthalpie change dH plotted against time. dH was calculated from the raw data of the ITC measurement by integration. It is evident that all of the injections result in similar enthalpy change, except for the first. Empty dots represent mask data points.

![Manual fitting of data](https://static.igem.wiki/teams/4208/wiki/project/results/220916-pctd-lbd-choline-analysis-screenshot-4.png )

Figure 16: Enthalpie change dH plotted against time. A curve was fitted to the data. The dissociation constant 2.6 µM and the concentrations of protein (35 µM) and ligand (490 µM) were set as constants.

#### Conclusion

To summarize this part of our project, we successfully expressed and purified a protein with the expected molecular weight of around 39.5 kDA, indicating that it is PctD-LBD.

The results of the ITC measurement did not allow us to quantify the binding of Choline to PctD-LBD. But the data we obtained allows the assumption that there is binding of choline to a small subset of protein that is functional. Whether this is indeed the case has to be examined in further experiments.

#### Future prospects

As it has already been shown in other publications that binding of Acetylcholine and Choline can be verified by ITC, the next step will be to confirm functionality of our purified protein e.g. through Blue Native PAGE or CD spectroscopy. Based on the results, the ITC can be repeated with adjusted concentrations of protein and ligand. Also, the purification protocol of PctD-LBD can be revised if the yield of functional protein is very low.

We expresssed the PctD-LBD in BL21(DE3) pLysS cells. In this strain, T7 RNA polymerase under control of lac UV5 promotor can be induced by IPTG. The T7 polymerase then transcribes target genes under control of the T7 promotor [(6)](#6). Additionally, the strain contains the pLysS plasmid which encodes T7 lysozyme, reducing the expression of T7 RNA polymerase and thus decreasing expression of the target gene in absence of IPTG [(7)](#7).

In the paper characterizing the PctD chemoreceptor, PctD-LBD expression was carried out in the BL21-AI strain, which has been engineered to reduce background expression by a different method. In this strain, the T7 RNA polymerase is expressed under the strictly controlled pBAD promotor which is activated by arabinose [(4)](#4) [(8)](#8).

The change of expression system compared to the literature might have contributed to misfolding of the protein, rendering it inactive. Expressing PctD-LBD in Bl21-AI cells could be carried out in an attempt to produce more functional protein.

After we verify the expressed protein is functional and binding of Choline and Acetylcholine can be shown by ITC, the last step will be to repeat the experiment with Anatoxin-a to answer the question whether Anatoxin-a also binds PctD.

### Testing of Anatoxin-a Biosensor

---

#### Introduction

As the description of the cloning and assembly of our biosensor shows, we created 2 potential candidates for our biosensor: VS1007 PHE and VS1007 PEH. Both candidates are based on the strain VS996 which lacks all chemoreceptors as well as EnvZ. VS1007 additionally contains the reporter plasmid pAM107 PompC-GFP expressing GFP under the PompC promotor. The biosensor candidates carry a second plasmid which contains the hybrid receptor - either the version PctD-HAMP-EnvZ (PHE) or the version PctD-EnvZ-HAMP (PEH). The hybrid receptor is under control of the PnahG promotor making its expression inducible with salicylic acid or acetylsalicylic acid.

To assure that our chimeric receptors and the signaling pathway leading to expression of GFP work as intended, we carried out the next experiments with the natural ligands choline and acetylcholine. Our plan was to test the biosensor with Anatoxin-a after the initial testing with choline and acetylcholine would have confirmed the functionality of the sensor.

First, we performed a growth experiment to assess the fitness cost of our plasmids. For this, we grew the constructed E. coli strains (containing 0, 1 or 2 plasmids) over a period of several hours. We measured the OD600 every hour to compare their growth curves.

Second, to get a first impression of the fluorescence of our potential biosensor, we imaged the different strains, incubated with no ligand, choline and acetylcholine with an epifluorescence microscope.

Finally, we chose the most promising candidate for our biosensor to measure the response to different concentrations of choline and acetylcholine over an extended period of time.

#### Results

 **Growth experiment**

The 2 biosensor candidates (VS1007 PHE, VS1007 PEH), as well as the reporter strain (VS1007) and the untransformed VS996 strain were grown in 10ml LB medium in 15 ml polypropylene tubes at 37°C and 200 rpm for 7h. The figure below (Fig. 17) shows the growth curves for each of the strains.

For each timepoint, 3 samples of each strain were measured. The growth curves are constructed from the mean values of these 3 measurements. The standard deviation is given for each timepoint as well.

![Growth curve](https://static.igem.wiki/teams/4208/wiki/project/results/igem-growth-curves-semilog-plot.png )

Figure 17: Growth curves of untransformed background strain (VS966), reporter strain (VS1007) and biosensor candidate strains (VS1007 PHE, VS1007 PEH). The measurements were terminated after 430 min (7h10min), at the beginning of the stationary phase.

 **Fluorescence Microscopy**

As we use GFP as a reporter, we expect to see a change in fluorescence of our E. coli cells upon binding of a ligand to the PctD ligand binding domain of our PctD-EnvZ chimeric receptor. Therefore, we used fluorescence microscopy to check whether our biosensor shows the intended behavior. choline and acetylcholine were used as ligands as they are natural substrates of PctD and should activate EnvZ signalling if the hybrid receptor is functional.

We used overnight cultures of our biosensor candidates and our reporter strains and induced the Pnahg rpomoter with acetylsalicylic acid. Approximately 1h before the imaging, the ligands (200µM) were added. We then used a fluorescence microscope to image each of the strains with and without ligand. The images are collected in the Figures 18-20.

The reporter strain VS1007 shows highly visible fluorescence under all 3 conditions. This indicates that the reporter plasmid promotor is leaky, resulting in GFP expression without stimulus.

![VS1007-microscopy](https://static.igem.wiki/teams/4208/wiki/project/results/microscopy-vs1007.svg)

Figure 18: Fluorescence microscopy images of the reporter strain VS1007 incubated with no ligand, acetylcholine (200µM) and choline (200µM). Fluorescence is visible in all panels.

VS1007, transformed with the plasmid containing PHE shows no fluorescence except for the fluorescence in E.coli. Adding of the ligand shows no visible effect.

![VS1007-phe-microscopy](https://static.igem.wiki/teams/4208/wiki/project/results/microscopy-vs1007-phe.svg )

Figure 19: Fluorescence microscopy images of the reporter strain VS1007 PHE incubated with no ligand, acetylcholine (200µM) and choline (200µM). No fluorescence except for the background fluorescence of E.coli is visible in all panels.

VS1007, transformed with the plasmid containing PHE, shows visible fluorescence under all 3 conditions. Because the VS1007 reporter strain shows leakage, some or all fluorescence from the VS1007 PEH strain stems from the reporter plasmid. To see differences in fluorescence of the 2 strains or under different conditions, the fluorescence output needs to be quantified.

![VS1007-peh-microscopy](https://static.igem.wiki/teams/4208/wiki/project/results/microscopy-vs1007-peh.svg )

Figure 20: Fluorescence microscopy images of the reporter strain VS1007 PEH incubated with no ligand, acetylcholine (200µM) and choline (200µM). Fluorescence is visible in all panels.

VS1007 PEH is the strain that seemed most promising for our biosensor, so we wanted to further test its function. We set up an experiment to quantitatively measure the fluorescence of the strain over time in response to different concentrations of ligand.

 **Plate reader experiment**

To quantify the fluorescence of our bacteria, we set up an experiment that allowed us to measure green fluorescence and absorbance at 600nm in a plate reader at the same time. We used the strain VS996 as controls and compared VS1007 and VS1007 PEH with no ligand and ligand in the following concentrations: 10 µM, 100 µM and 1000 µM. As in previous experiments, acetylcholine and choline were used as substrates.

We inoculated M9 medium for the different test conditions (15 in total) with overnight cultures of the three different strains (VS996, VS1007, VS1007 PEH) to a starting OD600 of 0.2. We induced the expression of the hybrid receptor with 4μM acetylsalicylic acid and added the ligands for the designated samples. Cells were incubated for 4h after induction and samples were taken in triplicates for measurement with the plate reader at 0h, 2h, 4h.

Analysis of absorption at 600 nm (Fig.21 ) shows that absorbance at 600 nm after 4 h is decreased in samples containing acetylcholine and choline in both VS1007 and VS1007 PEH. However, this is at least partly due to differences at the starting absorbance at 600 nm indicating growth is not strongly affected by the compounds. Absorbance at 600 nm is 2 to 3 times higher for VS996 compared to samples of VS1007 and VS1007 PEH which can again be attributed to a difference in the starting cell density.

![Abs600](https://static.igem.wiki/teams/4208/wiki/project/results/growth-vs1007-peh-allgrowthcurves.png)

Figure 21: The absorbance at 600nm plotted against the time t(h) for all the strains and conditions of the plate reader experiment (see legend).

Interestingly, for VS1007 PEH cells absorbance at 600 nm stayed almost level over the course of incubation indicating slower growth compared to the VS1007 cells. This effect does not depend on the addition of acetylcholine or cholin. Possibly, the induction of the hybrid receptor with acetylsalicylic acid placed a burden on the cells and impaired their growth. In the previous growth experiments, this effect could not be observed because the expression of the hybrid receptor had not been induced.

The figures below show the fluorescence normalized by the cell number (fluorescence/absorbance at 600 nm) over time.

First, we compared the background fluorescence of the different strains without added ligand (Fig. 22).

![Background Fluorescence](https://static.igem.wiki/teams/4208/wiki/project/results/testing-background-fluorescence.png  )

Figure 22: Fluorescence normalized by absorption at 600nm plotted against the time. Shown are the strains VS996, VS1007 and VS1007 PEH without any ligand added.

It is evident that VS966 shows very little fluorescence, probably only the autofluorescence of E. coli cells. Compared to that, the VS1007 reporter strain shows an approximately 3-fold increase in fluorescence. This is expected due to some leakage of the ompC reporter and consequential synthesis of GFP.

Interestingly, the VS1007 PEH strain shows an approx. 6-fold increase in fluorescence compared to the reporter strain. This indicates that our chimeric receptor activates GFP expression without stimulation by a ligand. This possibility is consistent with the literature. There are cases of chimeric chemoreceptors that activate expression of their reporter gene while inactive, and suppress it upon activation and vice versa [(9)](#9).

The Figures 23 to 26 summarize the normalized fluorescence of the VS1007 reporter strain when incubated with different concentrations of acetylcholine and choline. This strain does not have the plasmid containing the PEH gene, therefore no change of fluorescence upon adding any ligand is expected.

A paired Student´s T-test was performed for the data points of t = 4h for all pairings. The p-values p can be found in the section “p-values”. Significant results (p < 0.05) are indicated by brackets and asterisks in the following figures.

As expected, for addition of choline no significant changes are observed. When adding acetylcholine, the difference between 0µM and 100µM and between 100 µM and 1000 µM are considered significant. But for the first pair, increase of acetylcholine results in increased normalized fluorescence, while the second pair shows the opposite effect. In line with our expectations, this indicates that we do not observe a real effect of acetylcholine here.

![VS1007-acetylcholine](https://static.igem.wiki/teams/4208/wiki/project/results/testing-vs1007-acetylcholine.png)

Figure 23: Fluorescence normalized by absorption at 600nm plotted against the time. Shown is the strain VS1007 incubated with 0µM, 10µM, 100µM and 1000µM acetylcholine. Statistically significant differences at t = 4h are indicated by asterisks.

![VS1007-choline](https://static.igem.wiki/teams/4208/wiki/project/results/testing-vs1007-choline.png )

Figure 24: Fluorescence normalized by absorption at 600nm plotted against the time. Shown is the strain VS1007 incubated with 0µM, 10µM, 100µM and 1000µM choline. There are no statistically significant differences at t = 4h.

Next, the normalized fluorescence of the VS1007 PEH strain when incubated with different concentrations of acetylcholine and choline was compared. Based on the results for the samples with no added ligand, we expect to see a reduction in fluorescence over time. This reduction over time is expected to be more pronounced at increasing ligand concentrations. This is because based on our results we hypothesize that our chimeric chemoreceptor suppresses GFP expression upon ligand binding.

For most test conditions, the relative fluorescence decreases gradually over time. Only for the test condition with 100 µM choline, the fluorescent is increased after 2h of induction and then decreases below the initial level after 4h.
Also, an increase of normalized fluorescence (t = 4h) between 0µM and 1000µM, as well as between 10µM and 1000 µM acetylcholine is observed. Paired t-test revealed that this result can be classified as significant. Adding to the high variance across the samples and the non-uniform trend between samples, this suggests that the observed effect is due to statistical fluctuations instead of a biological effect.

![VS1007-peh-acetylcholine](https://static.igem.wiki/teams/4208/wiki/project/results/testing-vs1007-peh-acetylcholine.png )

Figure 25: Fluorescence normalized by absorption at 600nm plotted against the time. Shown is the strain VS1007 PEH incubated with 0µM, 10µM, 100µM and 1000µM acetylcholine. Statistically significant differences at t = 4h are indicated by asterisks.

![VS1007-peh-microscopy](https://static.igem.wiki/teams/4208/wiki/project/results/testing-vs1007-peh-choline.png )

Figure 26: Fluorescence normalized by absorption at 600nm plotted against the time. Shown is the strain VS1007 PEH incubated with 0µM, 10µM, 100µM and 1000µM choline. Statistically significant differences at t = 4h are indicated by asterisks.

For addition of choline, all pairings but 2 showed a significant difference in normalized fluorescence after 4 hours of incubation (Fig. 26). The higher the choline concentration, the higher the normalized fluorescence. Again, high variances across samples can be observed.
Taken together with the results for acetylcholine, this could indicate a biological effect. But opposed to our hypothesis, higher acetylcholine or choline concentrations resulted in higher normalized fluorescence.

#### Conclusion

We transformed the VS1007 reporter strain with plasmids that contain 2 different variants of a PctD-EnvZ chimeric chemoreceptor. We were able to show that our new strains are not impaired in their growth.

We showed fluorescence in the VS1007 and VS1007 PEH strains under the fluorescence microscope, but we could not show any effect of choline or acetylcholine on our biosensor candidate. Therefore, we sought to quantify the effect of different concentrations of choline and acetylcholine on VS1007 PEH by plate reader experiments.

In those experiments we showed that VS1007 had low background fluorescence. However, for VS1007 PEH we observed high fluorescence indicating active EnvZ signalling in the absence of ligands. Assuming a functional hybrid receptor, we would expect a ligand concentration dependent decrease in fluorescence over time. However, both ligands show an increase fluorescence for choline and acetylcholine compared to the samples where no ligand was added.

As both choline and acetylcholine show a trend to increase fluorescence with increasing concentration after 4h of incubation, an effect of those substrate on our biosensor can be suspected.

#### Future prospects

To further test the function of our biosensor candidate VS1007 PEH, the next step will be to repeat the plate reader experiment described above over an extended period of time with more biological replicates to validate the results. If an effect of choline and acetylcholine is verified, the experiments then could be repeated with Anatoxin-a.

If the biosensor shows low affinity to Anatoxin-a, or weak binding is shown in ITC experiments, the next step will be to increase the affinity of PctD by directed evolution. Then the fluorescence response of the new biosensor strain to binding of Anatoxin-a needs to be quantified, similar to the experiments described above.

After a functional Anatoxin-a biosensor is obtained, a distributable form of the biosensor can be generated. To be usefull for testing facilities, a testing kit for water samples would be the best option. To further optimize this testing kit, the fluorescence reporter could be changed to RFP, so the signal can easily be distinguished from the autofluorescence of chlorophyl. Another improvement would be to broaden the range of cyanotoxins the biosensor can detect.

## References

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1. Verma N, Qu X, Trozzi F, Elsaied M, Karki N, Tao Y, Zoltowski B, Larson EC, Kraka E. SSnet: A Deep Learning Approach for Protein-Ligand Interaction Prediction. Int J Mol Sci. 2021 Jan 30;22(3):1392. doi: 10.3390/ijms22031392.
2. SeeSAR version 12.1.0; BioSolveIT GmbH, Sankt Augustin, Germany, 2022, www.biosolveit.de/SeeSAR
3. H. Bekker, H.J.C. Berendsen, E.J. Dijkstra, S. Achterop, R. van Drunen, D. van der Spoel, A. Sijbers, and H. Keegstra et al., “Gromacs: A parallel computer for molecular dynamics simulations”; pp. 252–256 in Physics computing 92. Edited by R.A. de Groot and J. Nadrchal. World Scientific, Singapore, 1993.
4. Matilla et al. Chemotaxis of the Human Pathogen Pseudomonas aeruginosa to the Neurotransmitter Acetylcholine. mBio. 2022 Apr 26;13(2):e0345821. doi: 10.1128/mbio.03458-21.
5. Christensen VG, Khan E. Freshwater neurotoxins and concerns for human, animal, and ecosystem health: A review of anatoxin-a and saxitoxin. Sci Total Environ. 2020 Sep 20;736:139515. doi: 10.1016/j.scitotenv.2020.139515.
6. Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113-30. doi: 10.1016/0022-2836(86)90385-2.
7. Studier FW. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol. 1991 May 5;219(1):37-44. doi: 10.1016/0022-2836(91)90855-z.
8. Bhawsinghka N, Glenn KF, Schaaper RM. Complete Genome Sequence of Escherichia coli BL21-AI. Microbiol Resour Announc. 2020 Mar 5;9(10):e00009-20. doi: 10.1128/MRA.00009-20.
9. Luu RA, Schomer RA, Brunton CN, Truong R, Ta AP, Tan WA, Parales JV, Wang YJ, Huo YW, Liu SJ, Ditty JL, Stewart V, Parales RE. Hybrid Two-Component Sensors for Identification of Bacterial Chemoreceptor Function. Appl Environ Microbiol. 2019 Oct 30;85(22):e01626-19.

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