iGEM 2021 — Team Tuebingen

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# Team:Tuebingen/Notebook

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* [Home](https://2021.igem.org/Team%3ATuebingen)
* Project
  + [Description](https://2021.igem.org/Team%3ATuebingen/Description)
  + [Engineering](https://2021.igem.org/Team%3ATuebingen/Engineering)
  + [Experiments](https://2021.igem.org/Team%3ATuebingen/Experiments)
  + [Model](https://2021.igem.org/Team%3ATuebingen/Model)
  + [Results](https://2021.igem.org/Team%3ATuebingen/Results)
  + [Notebook](https://2021.igem.org/Team%3ATuebingen/Notebook)
* Human Practices
  + [Human Practices](https://2021.igem.org/Team%3ATuebingen/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3ATuebingen/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ATuebingen/Partnership)
  + [Communication](https://2021.igem.org/Team%3ATuebingen/Communication)
  + [Implementation](https://2021.igem.org/Team%3ATuebingen/Implementation)
* Team
  + [Team](https://2021.igem.org/Team%3ATuebingen/Team)
  + [Attributions](https://2021.igem.org/Team%3ATuebingen/Attributions)
* Parts
  + [Parts](https://2021.igem.org/Team%3ATuebingen/Parts)
  + [Contribution](https://2021.igem.org/Team%3ATuebingen/Contribution)
* [Plant Special Prize](https://2021.igem.org/Team%3ATuebingen/Plant)

Go To

[January](#Jan)
[February](#Feb)
[March](#Mar)
[April](#Apr)
[May](#May)
[June](#Jun)
[July](#Jul)
[August](#Aug)
[September](#Sep)
[October](#Oct)

# Notebook

On this page, you can see a summary of what we did each day/week over the course of the year to make our project happen. As it is sometimes hard to keep track of everything, this page is meant as a high-level overview.

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# January

| 1st | 2nd | 3rd | 4th | 5th | 6th | 7th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |
| 8th | 9th | 10th | 11th | 12th | 13th | 14th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |
| 15th | 16th | 17th | 18th | 19th | 20th | 21st |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  | Kick-off meeting |  |  |
| 22nd | 23rd | 24th | 25th | 26th | 27th | 28th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  | First regular team meeting (weekly) |  |  |
| 29th | 30th | 31st |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |

# February

| 1st | 2nd | 3rd | 4th | 5th | 6th | 7th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | Wiki & Design Meeting: Planning of Design Theme, Portfolio, and Website |  | Finance Introduction Meeting: Mentors from last year introduced us into our responsibilities and gave us useful advice. |  |  |  |  |
| 8th | 9th | 10th | 11th | 12th | 13th | 14th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  | Wetlab: first inventory of lab consumables from former iGEM teams | Wiki & Design, Drylab Meeting with Elias from last year’s team: Tips for building the Website and Wiki, Tips for Work in the Drylab |  |  |  |  |
| 15th | 16th | 17th | 18th | 19th | 20th | 21st |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  | Finance: communication with old team members to organize the team bank accounts | Wiki & Design Meeting with Lukas from previous iGEM Tuebingen teams: Tips on mentor search, Website building, and Wiki building |  |  |  |
| 22nd | 23rd | 24th | 25th | 26th | 27th | 28th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  | Project finding day | We got in contact with our university bank account managers |  |

# March

| 1st | 2nd | 3rd | 4th | 5th | 6th | 7th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |
| 8th | 9th | 10th | 11th | 12th | 13th | 14th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Wiki & Design Meeting: Creating the Portfolio using our sponsored Affinity Suite and further discussions about the Portfolio; Website Hosting discussion |  |  |  |  |  |  |  |
| 15th | 16th | 17th | 18th | 19th | 20th | 21st |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | Wiki & Design Meeting: Working on the Portfolio and Design Theme | Meeting with Prof. Schäffer |  | Meeting with Prof. Forchhammer |  | Social media meeting: kick-off & brainstorming about plans for our different social media channels |  |
| 22nd | 23rd | 24th | 25th | 26th | 27th | 28th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  | Meeting with Prof. Groß: Questions about genome mining |  |  | Finance meeting: Brainstorming about sponsoring. Discussion with mentors about raising money. | Social media meeting: introduction into Affinity software for editing our design scheme |  |
| 29th | 30th | 31st |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |

# April

| 1st | 2nd | 3rd | 4th | 5th | 6th | 7th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  | HP Meeting: Brainstorming of general project ideas | Finance Meeting: Application for Promega grant; List of all possible sponsors |  |  |
| 8th | 9th | 10th | 11th | 12th | 13th | 14th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  | Drylab Meeting: Brainstorming ideas and searching for local experts     HP Meeting: Podcast Conceptualisation I | Meeting with student council regarding sponsoring for registration fee | HP Meeting: Podcast Conceptualisation II |  |  |
| 15th | 16th | 17th | 18th | 19th | 20th | 21st |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Finance Meeting: Application for sponsorship by the student council was accepted; Created excel sheet to keep track pf expenses; Created Skype account for receiving calls |  |  |  |  | Social media Meeting: internal organisation, responsibilities for the different social media channels |  |  |
| 22nd | 23rd | 24th | 25th | 26th | 27th | 28th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Wiki & Design: Working on our Website     Finance Meeting: Contact to biotechnology companies for sponsorship |  |  | Drylab Meeting with Elias from last year's team, he gave us tips on genome mining, molecular dynamics simulations and Drylab collaboration ideas     HP Meeting: Getting in contact with Wüstewelle (radio channel) as well as Podcast Conceptualisation III |  | Meeting with Prof. Groß: Questions about genome mining |  |  |
| 29th | 30th |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Finance Meeting: IDT registration; Discussion of the ordering list. | Drylab Meeting with Prof. Ziemert: Questions about genome mining and our project in general |  |  |  |  |  |  |

# May

| 1st | 2nd | 3rd | 4th | 5th | 6th | 7th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  | Finance meeting: Created first list of laboratory material to order. |  |  |  |
| 8th | 9th | 10th | 11th | 12th | 13th | 14th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  | Drylab Meeting: Beginning work on Hidden Markov Models and Translating Genomes, further research of possible cyclotide candidates, and finding previous iGEM teams who did similar things |  | Finance meeting: phone calls sponsors; Contacted university administration for bank account. Conceptualisation of further sponsoring strategies. Application of second Promega grant.     HP Meeting: Meeting with Team Ioaninna I |  | German meet-up organized by iGEM Bonn & Kaiserslautern |  |
| 15th | 16th | 17th | 18th | 19th | 20th | 21st |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  | Social media meeting: talk about possible collaborations, planning of thank you posts for sponsoring     HP Meeting: Scheduling Podcast Episodes | Finance Meeting: Planning of Meeting with the company brand. Organisation of applications for other iGEM grants. |  |
| 22nd | 23rd | 24th | 25th | 26th | 27th | 28th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  | Drylab: Collecting further HMM profiles from sites such as PFAM, beginning genome mining | HP Meeting: Conceptualisation and creation of the Podcast Intro | HP Meeting: Elias and Erik try to explain Biology to Kira |  |
| 29th | 30th | 31st |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  | Video: Kick-off meeting |  |  |  |  |  |

# June

| 1st | 2nd | 3rd | 4th | 5th | 6th | 7th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | Drylab Meeting: Looking into the options of using Prosite profiles for genome mining, further work on HMMs and genome mining     HP Meeting: Podcast Description, Logo Design and finalization of the Intro | Meeting with iGEM Ioannina     Finance Meeting: Staying in contact with sponsors |  | Social media sub team: team building movie night |  |  |  |
| 8th | 9th | 10th | 11th | 12th | 13th | 14th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Finance Meeting: Impact grand, Contacted Roche, Twist, Benchling and Corning | Drylab Meeting: Keeping it up with genome mining |  | Meeting with Alex Grundmann from Twist Bioscience to talk about sponsoring possibilities |  |  |  |  |
| 15th | 16th | 17th | 18th | 19th | 20th | 21st |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | HP Meeting: Setting tasks and roles for the Podcast, solving problems in the concept and preparing the next interview | Safety instruction for the Forchhammer lab     Finance Meeting: Contacted Microsynth for sequencing labels | HP Meeting: Meeting with Team Ioaninna II | Organization Meeting: Plans for Sub teams     HP Meeting: Trial interview with our Team |  |  |  |
| 22nd | 23rd | 24th | 25th | 26th | 27th | 28th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Video: First day of shooting the promotion video (lab)     HP Meeting: Meeting to schedule radio interview | HP Meeting: Regular discussion, setting new tasks, brainstorming more ideas | Video: Second day of shooting the promotion video (botanical garden, forest, lab)     Finance Meeting: Impact grand, List of possible expenses; Ordering of gloves | HP: interview at Radio Wüste Welle | Drylab Meeting: Possible collaboration opportunity with iGEM Team IISER Kolkata; Thinking about how to edit cyclotides for different bioactivity; Molecular Dynamics Planning | Wiki & Design Meeting: Portfolio Printing; Postcard Collaboration Project from iGEM Team Düsseldorf; Wiki building begins | Video: Third day of shooting the promotion video (greenhouse); video editing begins |  |
| 29th | 30th |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | HP Meeting: Discussion about modifications for the podcast, reflection on last meetings brainstorm |  |  |  |  |  |  |

# July

| 1st | 2nd | 3rd | 4th | 5th | 6th | 7th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Wetlab: bring inventory up to date and stuff we need to our lab space     Finance meeting: Staying in contact with excellence cluster; List of chemicals     HP: "Real" Interview with our Team | HP Meeting: Briefing on Üner Kolukisaoglu for the Interview | Drylab Meeting: Refocusing of project towards cyclotide grafting; Idea of pre-screening Wetlab constructs through simulations |  | HP: Interview Üner Kolukisaoglu |  | Video meeting: editing of the promotion video     HP Meeting: Finalizing legal matters for the podcast, decision upon new HP Projects: field trip & panel discussion |  |
| 8th | 9th | 10th | 11th | 12th | 13th | 14th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Finance Meeting: Ordering postcards for collaboration     HP Meeting: Conceptualization of panel discussion in greater detail |  | Drylab Meeting: Homology Modeling of constructs; Learning about Molecular Dynamics with GROMACS |  | HP Meeting: Briefing on Nadine Ziemert for the Interview |  | HP Meeting: General organization, research for more interview partners as well as the field trip & panel discussion |  |
| 15th | 16th | 17th | 18th | 19th | 20th | 21st |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Wetlab: meeting with Prof. Brötz-Oesterhelt to talk about design and planning of the antimicrobial assays     Interview with MolecularCloud to present our project     Finance Meeting: Contacted Promega, IDT and Twist for laboratory materials |  | Drylab Meeting: Idea of coarse-grained simulations in conjunction to all-atom simulations     Team building: barbeque |  | HP Meeting: Interview Nadine Ziemert |  |  |  |
| 22nd | 23rd | 24th | 25th | 26th | 27th | 28th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| First day in the lab. We prepared a lot of media and buffers. | Second day in the lab. We finished our preparations. The lab work can start!     Social media meeting: planning of social media content for Promega silver sponsoring |  |  | Golden gate cloning level I (Assembly with BbsI-HF and transformation in competent E. coli)   Inserts cloned:CtAEP1 (AEP) and AEP precursor (prec.) | Overnight culture of Level 1 constructs from Monday | Social media: video filming and editing for Promega as part of sponsorship contract for the Silver Grant     Contact other possible sponsors, Ordering for Assays     Miniprep of clones of the overnight culture; control digest with BAmHI and control agarose gel electrophoresis of level 1 constructs |  |
| 29th | 30th | 31st |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sequencing of AEP and precursor constructs | Drylab Meeting with Dr. Thiel: Questions about Molecular Dynamics simulations; Supercomputer Access     Autoclaving of consumables and media     HP Meeting: Interview Andreas Dräger | Drylab Meeting: Getting access to supercomputer Cluster from Dr. Thiel; Planning of Molecular Dynamics simulations; Building of structures     Social media: video editing for iJET collaboration and sending project information to iGEM Aix-Marseille for collaboration (guess the project name from emoticons)     Team building: sports day with swimming, beach volleyball and table tennis |  |  |  |  |  |

# August

| 1st | 2nd | 3rd | 4th | 5th | 6th | 7th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | First meeting for writing of a review for the journal initiative from iGEM Maastricht     Cloning level II: assemble AEP (with HA tag, 35S promoter, 35S terminator), the cyclotide precursor (with Myc-tag, 35S promoter, 35S terminator) and GFP (with 35S promoter, 35S terminator) into an LII vector backbone by Golden Gate reaction and transform the LII plasmids into E. coli (Erik, Teresa, Mirjam)     HP Meeting: Meeting Team Taiwan I | Cloning level I: clone the synthesized AMP fragments into an LI vector backbone by Golden Gate cloning and transform them into E. coli;   pick colonies from the level II vector transformation for overnight cultures (Erik, Teresa, Mirjam)     HP Meeting: Maastricht Journal initiative meeting I | Miniprep of the LII cultures, control digest and agarose gel electrophoresis, send the plasmids for sequencing;   cloning level III: assemble level II AEP, GFP and cyclotide precursors with promoters, terminators and tags into one level III vector: GFP and precursor for 2in1 constructs and AEP, GFP and precursor for 3in1 constructs;   pick colonies from the level I vector transformation for overnight cultures (Erik, Teresa, Mirjam)     Drylab Partnership Meeting with Kolkata: Kolkata will help us build our simulations, we will run them using our supercomputer access     HP Meeting: Panel discussion meeting, working out deadlines and structur | Drylab Meeting with Dr. Thiel: Questions about supercomputer BinAC     Miniprep of the LI cultures, control digest and agarose gel electrophoresis, send the plasmids for sequencing;   pick colonies from the level III vector transformation for overnight cultures (Erik, Mirjam)     Finance Meeting: Excellence Cluster | Miniprep of the LIII cultures, control digest and agarose gel electrophoresis, control PCR send the plasmids for sequencing (Erik, Anton)     Meeting for review writing (journal initiative iGEM Maastricht) | Drylab Meeting: Talking about what exactly our Molecular Dynamics systems should look like |  |
| 8th | 9th | 10th | 11th | 12th | 13th | 14th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | Set precultures of E. coli containing level III 3in1 and 2in1 constructs for Midiprep (Anton, Erik) | Midiprep of 3 colonies for each 3in1 and 2in1, control digest of the purified plasmids, send plasmids for sequencing with multiple primers (Mirjam, Teresa, Erik) | Golden Gate cloning level IV: clone c\_blank, c\_CHEN\_1 and c\_CHEN\_6 into level III 3in1 and 2in1 vectors, Transformation of competent E. coli (Teresa, Erik)     HP Meeting: General discussion, contacting Experimenta Heilbronn for a possible collaboration in the panel discussion | Pick colonies and set overnight cultures (Mirjam) | HP Meeting: Meeting with Thomas Potthast; Podcast details and explanation | Drylab Meeting: Further learning of Molecular Dynamics simulations, including coarse-grained and all-atom |  |
| 15th | 16th | 17th | 18th | 19th | 20th | 21st |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | Finance Meeting: Organization of meeting with contact of experimenta; Checked bank status | We transformed Agrobacteria with constructs: LIII 3in1 14, LIII 3in1 3, LIII 3in1 16, LIII 2in1 3, LIII 2in1 16. (Mirjam, Erik) | HP Meeting: General discussion of podcast, field trip and panel discussion | From the overnight cultures we picked 2 colonies per construct and inoculated those. (Mirjam, Erik) | We infiltrated 2 plants per construct and a negative control with AS medium. (Teresa, Catia, Mirjam)     HP Meeting: first meeting with Dr. Robert Friedrich from the experimenta for a possible collaboration in the panel discussion | Drylab Meeting: Beginning work on supercomputer BinAC; Developing collaboration with iGEM Team CCU Taiwan |  |
| 22nd | 23rd | 24th | 25th | 26th | 27th | 28th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Finance Meeting: Text for bank company; Ordering for excellence cluster | We checked the GFP expression under the fluorescence microscope. |  | We prepared media for the Assays and extracted the 3in1 14, 3in13 and the untransformed constructs from the leaves. (Mirjam, Catia)     HP Meeting: meeting with experimenta II   Subsequent discussion   General discussion, trying to win the Dieter Schwarz Stiftung as a sponsor in exchange for working with experimenta | We carried out the Assays, to determine the effect of our crude extract on B. subtilis and E. coli in different media (BHI, MH) and different concentration. (Teresa, Alicia, Catia) | We checked the results of the forementioned assay by checking the absorbance under light of 600nm (Alicia) | Drylab Meeting: Running first simulations from iGEM Team IISER Kolkata     Giant Jamboree fee paid |  |
| 29th | 30th | 31st |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | Erik prepared buffers for Wetlab     Received consumables from biotech companies; contacted bank account manager | HP: Interview Thomas Potthast |  |  |  |  |  |

# September

| 1st | 2nd | 3rd | 4th | 5th | 6th | 7th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| We extracted the infiltrated leaves that were prepared on the 20th august (3in1 14, 3in1 16 and untransformed samples). We then used these extracts to purify them via the included His-tag. Unfortunately, we could not measure proteins in the purified extracts. (Mirjam, Teresa, Erik, Alicia) | Since we could not detect proteins the day before, we repeated the his-tag purification with the flow-through from the day before. We then concentrated the elution fractions with dialysis and measured again the protein content of our solution. (Erik, Teresa, Alicia) | We quantified the amount of GFP in the cellular crude extract and flow-through (Teresa, Erik) |  |  | Tricine SDS-PAGE and western blot, set overnight cultures of agrobacteria with 3in1 c\_blank, c\_CHEN\_1 and c\_CHEN\_6 (Mirjam, Teresa, Erik)     HP Meeting: Collaboration: Interview with Team CCU Taiwan | Continue with the western blot, set new overnight cultures of agrobacteria with 3in1 c\_blank, c\_CHEN\_1 and c\_CHEN\_6, protein quantification assay (Mirjam, Teresa, Erik) |  |
| 8th | 9th | 10th | 11th | 12th | 13th | 14th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| HP Meeting: Discussion about experimenta, podcast, collaboration Team Taiwan | HP Meeting: Meeting with experimenta III, getting advice on the panel discussion, working on the collaboration |  |  |  | Drylab Meeting: Discussion of Presentation Video Ideas; Running coarse-grained simulations for force-analysis; Collaboration CCU Taiwan Structure Prediction     Protein extraction of constructs 3, 14 and 16 using 12 leaves of each plant was done.   Protein extraction of constructs 3, 14 and 16 using 12 leaves of each plant was done.   We purified all 3 constructs via His tag affinity purification and dialysis and quantified protein concentrations of all constructs afterwards. (Erik, Inga, Julius)   We also carried out cloning of Level III vectors (constructs 9 and 18) with subsequent transformation of E. coli. (Erik, Adrián, Laura, Dennis, Julius, Inga) | Wiki & Design Meeting: Writing of Wiki pages delegated to sub teams, and further planning regarding Wiki     We prepared samples for the SDS-PAGE and inoculated 2 of the colonies which have grown over night. An inhibition zone assay using our purified protein suspensions was carried out. (Laura, Dennis, Adrián, Mirjam, Inga) |  |
| 15th | 16th | 17th | 18th | 19th | 20th | 21st |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| We extracted and purified plasmid DNA from the overnight cultures using the Monarch Plasmid Miniprep Kit from NEB.   We assembled Level III vectors using the purified plasmid DNA of constructs 9 K1, 9 K2, 18 K1 and 18 K2 and transformed them into E. coli.   As yesterday's inhibition zone assays did not work, we repeated them with E. coli und B.subtilis. We tested different synthetic peptides as well as crude extracts or purified solutions of our grafted peptides.   We also used the purified protein solutions for SDS-PAGE with subsequent instant blue staining or Western Blot. (Laura, Dennis, Dilara, Adrián)     HP Meeting: Finishing draft expose and invitation mails for the panel discussion | As the inhibitions zone assay of yesterday yielded no significant results, we repeated them with higher concentrations.   Level I plasmids were digested with HindIII and analyzed gel electrophoresis. Overnight cultures of Level III plasmids were inoculated.   We continued yesterday's Western Blot and carried out the detection as well.   For purification, we carried out an acetone precipitation for several crude extracts as well already purified protein solutions. (Laura, Dennis, Dilara, Adrián, Erik, Julius, Inga)     HP Meeting: Meeting concerning the field trip to icon genetics/nomad bioscience in Halle | We extracted and purified plasmid DNA of yesterday's overnight cultures using the Monarch Plasmid Miniprep Kit from NEB. Afterwards, a control digest with subsequent gel electrophoresis was carried out.   As yesterday's Western Blot showed no significant signals, the membrane was washed and incubated with primary antibody over the weekend at 4°C. (Erik, Inga)     Taking team pictures for Wiki | Drylab Meeting: Further progress on coarse-grained umbrella simulations; Running and Results of Molecular Docking; Further work with IISER Kolkata | Social media meeting: planning for Wiki, other plans for possible interviews | We finished our western blot and purified our constructs via His-tag purification.   Replication of gel of control digestion of construct LIII 3in1 9 &18. | Agrobacteria transformation with 3in1 9 & 18.   SDS-PAGE and Western blot of constructs 3in1 3 & 14.     HP: Field trip to icon genetics/ nomad bioscience in Halle |  |
| 22nd | 23rd | 24th | 25th | 26th | 27th | 28th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Continuation of yesterday's labwork.     HP: Field trip to icon genetics/ nomad bioscience in Halle | Drying of leaves infected expressing 2in1 & 3in1 16 and p19. Overnight culture of transformed Agrobacteria.     HP: Field trip to icon genetics/ nomad bioscience in Halle | Extraction of 2in1 & 3in1 16 and p19 constructs (according to Poon et al., 2018).     Taking team pictures for Wiki | Drylab Meeting: Finishing of Taiwan Structures, starting simulations |  | Wetlab: Extraction of proteins from plant material directly with Lämmli buffer (untransfected, p19 control, constructs 2in1 3&16, 3in1 3,14&16)   SDS-PAGE with these samples plus from the extraction from 24th September, Coomassie staining of one gel, start of Western Blot with 1. Ab anti-His-Tag   Transformation of A. tumefaciens with plasmid with construct 3in1 9 | Wetlab: Finishing and detection of Western Blot, repeating of SDS-PAGE with same samples, Coomassie staining of one gel, start of two Western Blots with 1. Ab anti-His-Tag and 1. Ab anti-HA-Tag   microdilution assay for antimicrobial activity testing with our synthesized reference peptides, CHEN and KR-12 |  |
| 29th | 30th |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Wetlab: Finishing and detection of Western Blots, read out of assay from yesterday     Wiki Writing Day (the team came together and continued working on the Wiki pages)     HP Meeting: General discussion, solving questions concerning Wiki and panel discussion | Wetlab: Inoculation of overnight cultures of A. tumefaciens with colonies of constructs 3in1 9, 3in1 18 and 3in1 16 |  |  |  |  |  |  |

# October

| 1st | 2nd | 3rd | 4th | 5th | 6th | 7th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Wetlab: Infiltration of N. benthamiana plants with O.N. cultures from the day before and p19 vector as control | Wiki Writing Day 2 (the team came together and continued working on the Wiki pages)     Drylab Meeting: Recap of coarse-grained simulations     HP Meeting: Meeting with experimenta Heilbronn IV |  | Wetlab: harvesting of plants from Friday, fluorescence microscopy of whole plants to check infiltration efficiency, brightfield and GFP filter microscopy of single infiltrated leaves, protein extraction from plant tissue with new extraction buffer (Teresa, Erik)   Western Blot: re-incubate membranes from last week with freshly prepared primary antibody solutions (anti-His-Tag and anti-HA-Tag)   HP Meeting: Contacting the Dieter Schwarz Stiftung | Wetlab: SDS removal via acetone precipitation, microdilution assay for determination of antimicrobial activity of samples from 4th October (Alicia, Erik, Catia) | HP Meeting: General discussion and recap     Wetlab: SDS-PAGE and subsequent either Coomassie staining or western blot with samples extracted with the new extraction buffer from the 4th, read out of the antimicrobial assays from the 5th (Catia, Erik, Inga) | Wetlab: washing and detection of the western blots from the 6th (Alicia, Catia)     HP Meeting: Meeting with experimenta V |  |
| 8th | 9th | 10th | 11th | 12th | 13th | 14th |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Wetlab: extraction of protein from gel slices cut out of from SDS-PAGE gels for further analysis via MALDI-TOF/MS (Erik, Inga) | Drylab Meeting: Analysis of Membrane Simulations; Recap Meeting Kolkata | Video Meeting: Presentation Video, first day of shooting | Video Meeting: Presentation Video, second day of shooting     HP: Interview Heike Brötz-Oesterhelt     Wetlab: MALDI-TOF/MS experiments (Erik, Teresa) |  |  |  |  |
| 15th | 16th | 17th | 18th | 19th | 20th | 21st |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  | HP: Meeting with experimenta (VI) as preparation for panel discussion |  | HP: panel discussion in cooperation with experimenta |  |  |

### About Us

We are the iGEM Team Tuebingen, a group of motivated students who are working on creating a fast screening platform for stabilized peptides. We are aiming to provide a system that gives everyone the ability to stabilize peptides such as antimicrobial peptides to create better medical agents.

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### Contact Us

#### team@igem-tuebingen.de

#### +49 7071 9189 745

---

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# Team:Tuebingen/Engineering

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* [Home](https://2021.igem.org/Team%3ATuebingen)
* Project
  + [Description](https://2021.igem.org/Team%3ATuebingen/Description)
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  + [Experiments](https://2021.igem.org/Team%3ATuebingen/Experiments)
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  + [Human Practices](https://2021.igem.org/Team%3ATuebingen/Human_Practices)
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* [Plant Special Prize](https://2021.igem.org/Team%3ATuebingen/Plant)

Go To

[Design](#Design)
[Build](#Build)
[Test](#Test)
[Learn](#Learn)

# Engineering Success

Read about how we leveraged the engineering design cycle to change the direction of our project and make a better project.

---

# Design

## Wetlab

Following our decision to create a project on the resistance to antibiotics and the usage of some type of substitute as a solution for this problem, we started our designing process by brainstorming ideas about alternatives to antibiotics. We came across antimicrobial peptides (AMPs) which have proven to be effective against bacteria, viruses, fungi, and even cancer cells
[1](#1)
. These peptides can be naturally found in animals and plants, as they are part of the innate immune response
[2](#2)
. AMPs are a sophisticated way of fighting infections since compared to antibiotics, pathogenic bacteria have a far smaller likelihood of growing resistant against AMPs with time
[2](#2)
[3](#3)
. Deeper research into this topic showed us, that AMPs are a broad topic with many different AMP classes and origins. However, even if many AMPs are known, most of them are not very well characterized. This led us to the decision that we wanted to make a platform to create and test AMPs. However, in the laboratory and especially clinical use most AMPs have shown to be rather unstable and thus not suitable as a substitute to antibiotics
[4](#4)
[5](#5)
. We had to face this challenge and needed to come up with a strategy to increase the AMPs’ stability to use them
*in-vitro*
. A discussion with Prof. Groß gave us the idea of using cyclotides in our process. Cyclotides are plant-derived peptides with a cyclic structure. Cyclotides are known to be highly stable through their special structure which comprises disulfide bonds inside the circular structure of the peptides
[6](#6)
. However, further reading showed us, that the antimicrobial activity of cyclotides often is accompanied by hemolytic activity, which makes cyclotides unsuitable for medical use. This led us to the idea to integrate none-cyclotide AMPs, whose antimicrobial activity has been shown, into a none-hemolytic cyclotide scaffold to make these AMPs more stable and easier to handle. We decided to use Golden Gate cloning for generating the constructs since this is a preferably fast method, as described in more detail in our cloning
[results part](https://2021.igem.org/Team%3ATuebingen/Results)
. In general, we generated plasmids containing the AMP-cyclotide constructs, the green fluorescent protein (GFP), and the cyclizing enzyme asparagine endopeptidase (AEP). We designed a workflow to clone the constructs, express them in
*Nicotiana benthamiana*
, extract the cyclotides and purify them via an integrated His-tag and test their antimicrobial activity. We designed our workflow in a way to be a suitable platform for the fast and efficient testing of different AMPs.

## Drylab

### Design Iteration #1

Our first goal in Drylab was finding potential new antimicrobial peptides (AMPs) in the form of cyclotides. We first tried different methods of finding amino acid sequences like the ones we found in common databases and other resources.

It was important that the new findings contain the characteristic sequence of cysteine residues at predefined positions relative to each other, which are required to preserve the typical loop structures found in cyclotides.

Other residues between the cysteines should be allowed to vary.

### Genome Mining for New AMPs

Using search engines like BLAST provided by NCBI wasn’t fruitful, as its results only provided us with sequences already available to us.

This makes sense, as BLAST, by using a so-called seed and extend approach, splits up the sequence provided by the user into keywords and searches its database for sequences featuring similar keywords (called seeds).

The sequences containing those seeds are then further analyzed by comparing the amino acids surrounding the seeds to the input sequence. If the similarity score crosses a certain threshold, they are returned as search results. Therefore, BLAST doesn’t give particular importance to the cysteine residues that are important for the loop structures compared to other amino acids at other positions, which meant that BLAST wasn’t the suitable algorithm for our use case.

Soon afterward, we learned about Hidden Markov Models (HMM) and their use in bioinformatics to find similar sequences based on a multiple sequence alignment (MSA).

Using HMM in combination with plant genomes we expected to contain still undiscovered AMP sequences, we finally found promising results.

y using an MSA as input, HMMs evaluate each residue according to its frequency at specific positions of the MSA.

As our selection of cyclotides, when aligned properly, all have cysteine residues at the same position, the HMM favors amino acid sequences that are similarly structured regarding cysteine residues.

Other positions in the MSA, which are not as relevant for the loop structures, have more variation in amino acids, therefore allowing more variation at those positions in the search results.

After some research, we decided to use HMMER, a sequence homolog search tool using HMMs, provided by hmmer.org, using Cucurbitaceae, Fabaceae, Solanaceae, Rubiaceae, and Violaceae genomes as a database and the MSA of the amino-acid sequences of circulin B, cycloviolacin O2 as well as cyclopsychotride A as an HMM.

This approach finally gathered plenty of useful results.

### Design Iteration #2

Due to the hemolytic activity of cyclotides and the consequent risk for our Wetlab members, we decided to change our goal mid-project.

Instead of finding new AMPs and testing their antimicrobial activity, we decided to tackle the inherent stability issues of most non-cyclic AMPs by integrating (or grafting) their amino-acid sequence into the sequence of a cyclotide scaffold.

We expected the cyclic scaffold to stabilize the resulting construct while conserving its antimicrobial activity without potential harmful hemolytic activities.

For the Drylab part of the project, this meant that instead of finding new AMPs, we would now focus on testing potential cyclotide-AMP constructs before the Wetlab would start expressing and testing them manually.

Broken down, we had to find a method to test the stability of the construct as well as its interaction with a membrane.

After some research and meetings, it was clear we had to find a way to predict a tertiary structure from our amino-acid sequence, which we then had to use in simulations that allowed conclusions about the above-mentioned properties.

# Build

## Wetlab

Derived from the designing process, our experimental design had four major steps: cloning of our vectors, AMP expression in
*Nicotiana*
, peptide extraction and purification, and finally antimicrobial activity testing. We started with the cloning process in competent
*E. coli*
cells where we generated the desired plasmids, as described in more detail in the cloning
[results](https://2021.igem.org/Team%3ATuebingen/Results)
part. After purification of our
[final vectors](https://2021.igem.org/Team%3ATuebingen/Results)
, we transformed
*Agrobacteria*
with the respective plasmids for agroinfiltration. We used the agrobacteria to infiltrate the leaves of tobacco plants for transient protein expression. As we integrated the green fluorescent protein (GFP) into the plasmid, we could check the leaves for fluorescent signals as a first protein expression control. Thereafter, proteins were mechanically extracted by pestling under liquid nitrogen
[7](#7)
. Since our constructs were labeled by a His-tag, our next step after extraction was to purify the cyclotide with affinity purification. In order to gain higher concentrations, we use acetone precipitation. The final step in our project was to test the antimicrobial activity in different assay conditions.

## Drylab

### Predicting the Tertiary Structure

Right around the time we started working towards our new goal, AlphaFold 2.0, developed by DeepMind and EMBL-EBI, was released to the public.

Before its release, AlphaFold 2.0 made headlines regarding its then unmatched accuracy in predicting the fold and therefore tertiary structure of proteins in the Critical Assessment of Protein Structure Prediction (CASP) competition in November 2020.

As the capacity of our personal computers was limited, we had to use
[AlphaFold Colab](https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb#scrollTo=VzJ5iMjTtoZw)
, a simplified version of AlphaFold 2.0.

Most of the results looked promising, but all lacked the essential connection between the first and last amino acid.

![Predicted structure of a cyclotide lacking the bond between the first and last amino acid](https://static.igem.org/mediawiki/2021/5/53/T--Tuebingen--engineering_modded-cyc.png)

Figure 1: Predicted structure of a cyclotide lacking the bond between the first and last amino acid

We guess that AlphaFold 2.0 misses such a bond due to the rareness of proteins featuring a cyclic structure.

To fix this, we wrote a program that would cyclize the structures for our purposes (see
[Model](https://2021.igem.org/Team%3ATuebingen/Model)
).

# Test

## Wetlab

We integrated testing and controls steps in nearly every step of our protocol. At the beginning of our process, we tested whether the constructed plasmids were correctly expressed in
*E. coli*
. For this, we used restriction digest and sequencing of our purified vectors. Furthermore, selective pressure of different antibiotics allows only correct clones to grow and we used blue-white screening in some cloning steps, as described in the cloning
[results](https://2021.igem.org/Team%3ATuebingen/Results)
section. After knowing, that the cloning worked, we needed to test our next major step, the peptide expression. Therefore, in the next step, we needed to check if the protein expression of our plasmid was successful in the transfected tobacco leaves. We designed our vectors in a way to easily allow this testing. GFP was part of the plasmid, so we were able to examine the leaves under a fluorescent microscope and verified the fluorescence intensity. Only leaves showing green fluorescence were harvested for protein extraction and purification, as described in the Expression in
*Nicotiana*
[results](https://2021.igem.org/Team%3ATuebingen/Results)
section. Afterwards we checked with SDS-PAGE and western blot with an anti-His antibody whether our protein of interest, the cyclotide, was expressed. In the western blots, we used untransfected leaves as a negative control and a protein with His-tag as a positive control. For additional controls, testing the expression of GFP with an anti-GFP antibody or the expression of the AEP with an anti-HA antibody would also be possible. This could give us some hints if the proteins of our plasmid were expressed in the plant. In addition, we tested the antimicrobial activity of our constructs to determine if the AMPs that are incorporated into the cyclic structures are still functional. We used microdilution assays and radial diffusion assays to determine the antimicrobial properties. We conducted a series of experiments with different media since we were aware that activity can be hidden by the used media. For positive controls, we used the synthesized AMPs KR-12
and CHEN. In this way, we could determine if there is a change in their antimicrobial activity when incorporated into our cyclotide construct. In addition, we needed the control peptides to adapt the assays to our samples. We also included untransfected plants and plants transfected with p19 as negative controls. This was necessary to exclude that the leaves themselves have any antimicrobial activity. Moreover, we wanted to test the stability of our constructs since we expected them to be more stable compared to the single AMPs. However, due to lack of time, we could not do any stability analysis.

## Drylab

### Running simulations

To test the construct for stability and its capability to interact with the membrane, we had to run a molecular dynamics (MD) simulation.

No one on our team had any experience regarding simulations or in using Gromacs
[8](#8)
[9](#9)
[10](#10)
, our simulation software of choice.

However, we were able to reach out to several experts, including Dr. Thiel from the biomedical informatics department of the university hospital Tübingen, as well as team IISERK from Kolkata (see
[Partnership](https://2021.igem.org/Team%3ATuebingen/Partnership)
).

Each of them helped us greatly towards understanding more about MD and Gromacs specifically.

Thanks to Dr. Thiel, we also were granted access to the supercomputer BinAC in order to perform some of our simulations.

Generally speaking, the simulations take place within a virtual box containing the predicted protein structure and a so-called solvent (e.g., water molecules).

Those simulations can be performed at different levels of precision.

An all-atom simulation describes a simulation where the trajectory of each atom is being computed (which is computationally intensive). In contrast, a coarse-grained simulation sums up groups of atoms to single entities, for which the trajectories are being calculated (can even be computed on a consumer-grade computer).

Please refer to our experiment wiki entry for more general information about MD simulations using Gromacs (see
[Experiments](https://2021.igem.org/Team%3ATuebingen/Experiments)
).

In order to get the desired data about our constructs, we had several simulation setups:

* A coarse-grained simulation containing the construct and a membrane
* An all-atom simulation containing the construct and a membrane, provided by team IISERK from Kolkata

The dual setup was to get an idea about which construct performs best through coarse-grained simulations before running the all-atom simulations on BinAC which takes way longer.

![An all-atom model](https://static.igem.org/mediawiki/2021/c/cd/T--Tuebingen--engineering_all-atom-sys.png)

Figure 2: An all-atom model

![A coarse-grained model](https://static.igem.org/mediawiki/2021/3/30/T--Tuebingen--engineering_coarse-grained-sys.png)

Figure 3: A coarse-grained model

### Simulating Lipid to Protein Interaction

Apart from using Gromacs for simulation purposes, we also used AutoDock
[11](#11)
.

Given a protein (in our case one of our constructs) and a ligand (a phospholipid), AutoDock perceives their positioning relative to each other as “genes”: values describing their conformation, orientation, and translation which can undergo random mutations which may increase their “fitness”, a score representing the total interaction energy of ligand and protein.

Initially, multiple instances of the ligand are being generated, each with a different set of genes.

Based on their fitness score, their chance to “mate” with another instance and create a new instance with a genetic set derived from its parents is computed.

The offspring can undergo some random mutation, altering its positioning relative to the protein.

Through this procedure, instances with a bad fitness die out, while those with higher interaction energy replicate and have a chance to increase their score by mating with others further.
[12](#12)

Using AutoDock showed us which construct binds best to a phospholipid, and therefore which has the best chance to disrupt a membrane.

However, we also knew that it only shows the binding between a construct and a free phospholipid, not between the construct and a membrane.

This means that the simulated interaction can only happen once the construct has perforated the membrane.

To conclude which construct binds best to the membrane (or is best at initializing the perforation), the results from MD were more relevant.

[](https://static.igem.org/mediawiki/2021/8/8a/T--Tuebingen--model-chen_l1_complex.mp4)

Figure 4: c\_CHEN\_1 docked to the POPG phospholipid

# Learn

## Wetlab

We continuously had to adapt our project idea and workflow, due to new information and realizations from the lab. With new ideas, our design – build – test cycle had to start again and new experiments were performed in the lab until new results forced us to integrate new adaptations. When we started with the practical work, we were aware of the fact, that one usually needs more time than expected because of failure in experiments. However, the first experiments we did worked remarkably well. We never had issues with the Golden Gate cloning system and generated our plasmids very fast. However, after the first enthusiasm, we faced many challenges, while we performed our experiments in the lab. In general, we learned a lot about how to work in a team and what should be done if an experiment is not working or results are not conclusive. Good planning when starting with the lab work is essential because we needed to do as many experiments as possible in a short
time.

The cloning and expression in tobacco processes worked as planned. The first challenge we faced was that the extraction and purification process showed the desired results in neither the western blots nor the antimicrobial assays. We started extensive troubleshooting to determine what could be the problem and how we could make our system work, which is described in detail in the Extraction and Purification results (Verlinkung Troubleshooting results extraction and purification) section. In short, we discussed eight potential problems that might be responsible for our protocol failure: (A) Problems with the blotting procedure, (B, C) Expression of the peptide or the AEP in plants did not work, (D) Low expression levels, (E) Inefficient crushing of the leaves, (F) Unsuitable extraction buffer, (G) Problems with the His-tag recognition and (H) Binding of our peptide to plastic. After defining our potential problems and finding possibilities for testing if they
are a problem, we were able to exclude some of them, like problems in the blotting procedure by including a positive control. We noticed, that we could not directly test for some of our potential problems, like the expression of the peptide in plants, as all our testing strategies required a successful extraction beforehand. For other potential problems, we did extensive testing in the lab but were still not able to solve the problem. We considered the extraction buffer to be the most likely problem and tested four more buffer compositions, but none of them showed the desired results.

A highly important fact we learned was including controls. This is crucial for verifying the results. For example, in the first western blots, we did not include positive controls. This made the interpretation of results difficult since we were not sure if we had no protein of interest in the samples or if the antibody failed. Controls were also crucial for our assays since we needed a positive control to test if our assays were suitable for our AMPs, and negative controls to ensure that the tobacco extract itself did not possess any antimicrobial activity.

We also noticed how important it is, to find advisors that are experts in our field of studies. Our team did a lot of research and we read countless papers, but in the end, the advice and input we got from our PIs were essential to shape our work. As our project included different steps, we consulted different experts. Dr. Kolukisaoglu was our primary PI and helped to shape the whole project and was especially experienced in working with plants. Prof. Brötz-Oesterheld especially helped us with the antimicrobial assays. Prof. Groß allowed us to do the acetonitrile extraction in his lab, amongst other things. When our project took a new direction, we also looked for more experts that could help us with the new ideas. What also helped our project a lot was the excursion to Nomad, where we not only got ideas for the implementation of our project but also got advice on how to specifically change experiments in the lab. All in all, our project would not have been
possible without the input of so many experts and we want to thank all of them for their help.

When our lab work started, we also learned how essential good communication in the team is. Our Wetlab team included many members and we took turns in working in the lab. Therefore, experiments one person started were often continued by another person. Good documentation of the lab work was essential to make this possible. In our weekly Wetlab meetings we discussed the results from the last week and planned the next week, so everyone was up to date. However, when experiments did not work as planned, the people in the lab had to decide how to solve these problems. As our team members had different levels of experience in working in the lab and also a different level of knowledge about our project, it was important that other team members were available when severe problems came up. In the end, we really learned how to organize ourselves as a team.

To sum up, we had to tackle many issues regarding our project. Of course, we were not able to solve every single problem especially due to the lack of time. However, we did a lot of troubleshooting and discussion and found suitable solutions for some issues. In addition, we have several ideas about how the project could continue. And what is even more important, we as a team gained a lot of knowledge in the methods we used. We learned how to plan, execute and analyze experiments on our own, which is crucial to be a successful scientist. In addition, we trained our soft skills like organize ourself and others and working together as a team. The iGEM competition provides an opportunity to develop yourself further, especially outside your professional field. We, iGEM Team Tübingen, can proudly claim that every single team member seized this opportunity!

## Drylab

### Analysis of Gromacs Simulations

After finishing the simulations, we used VMD
[13](#13)
to analyze the constructs regarding their interaction with the membrane and learn from the data we collected. We analyzed the parameters Root Mean Squared Deviation, Solvent Accessible Surface, and Hydrogen Bonds (for more details, see
[Model](https://2021.igem.org/Team%3ATuebingen/Model)
).

# References

1
Harris, Frederick; Dennison, Sarah R.; Phoenix, David A. (2009): Anionic antimicrobial peptides from eukaryotic organisms. In Current protein & peptide science 10 (6), pp. 585–606. DOI: 10.2174/138920309789630589.

2
Boparai, Jaspreet Kaur; Sharma, Pushpender Kumar (2020): Mini Review on Antimicrobial Peptides, Sources, Mechanism and Recent Applications. In Protein and Peptide Letters 27 (1), pp. 4–16. DOI: 10.2174/0929866526666190822165812.

3
Zhu, Meng; Liu, Peng; Niu, Zhong-Wei (2017): A perspective on general direction and challenges facing antimicrobial peptides. In Chinese Chemical Letters 28 (4), pp. 703–708. DOI: 10.1016/j.cclet.2016.10.001.

4
Koehbach, Johannes; Gani, Jurnorain; Hilpert, Kai; Craik, David J. (2021): Comparison of a Short Linear Antimicrobial Peptide with Its Disulfide-Cyclized and Cyclotide-Grafted Variants against Clinically Relevant Pathogens. In Microorganisms 9 (6), p. 1249. DOI: 10.3390/microorganisms9061249.

5
Gunasekera, Sunithi; Foley, Fiona M.; Clark, Richard J.; Sando, Lillian; Fabri, Louis J.; Craik, David J.; Daly, Norelle L. (2008): Engineering stabilized vascular endothelial growth factor-A antagonists: synthesis, structural characterization, and bioactivity of grafted analogues of cyclotides. In Journal of medicinal chemistry 51 (24), pp. 7697–7704. DOI: 10.1021/jm800704e.

6
Craik, David J. (2010): Discovery and applications of the plant cyclotides. In Toxicon : official journal of the International Society on Toxinology 56 (7), pp. 1092–1102. DOI: 10.1016/j.toxicon.2010.02.021.

7
Poon, Simon; Harris, Karen S.; Jackson, Mark A.; McCorkelle, Owen C.; Gilding, Edward K.; Durek, Thomas et al. (2018): Co-expression of a cyclizing asparaginyl endopeptidase enables efficient production of cyclic peptides in planta. In J Exp Bot 69 (3), pp. 633–641. DOI: 10.1093/jxb/erx422.

8
E. Lindahl, B. H. (2001). GROMACS 3.0: A package for molecular simulation and trajectory analysis. Journal of Molecular Modeling, 306-317.

9
H. Bekker, H. B. (1993). Gromacs: A parallel computer for molecular dynamics simulations. Physics computing, 252–256.

10
H.J.C. Berendsen, D. v. (2001). GROMACS: A message-passing parallel molecular dynamics implementation. Computer Physics Communications, 306-317.

11
Morris, G. M. (2009). Autodock4 and AutoDockTools4: automated docking with selective receptor flexiblity. Journal of Computational Chemistry, 2785-9271.

12
Garrett M. Morris, D. S. (1999). Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. Journal of computational chemistry, 1639-1662.

13
William Humphrey, A. D. (1996). VMD - Visual Molecular Dynamics. Journal of Molecular Graphics, 33-38.

### About Us

We are the iGEM Team Tuebingen, a group of motivated students who are working on creating a fast screening platform for stabilized peptides. We are aiming to provide a system that gives everyone the ability to stabilize peptides such as antimicrobial peptides to create better medical agents.

### Follow Us

### Contact Us

#### team@igem-tuebingen.de

#### +49 7071 9189 745

---

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# Team:Tuebingen/Experiments

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* [Home](https://2021.igem.org/Team%3ATuebingen)
* Project
  + [Description](https://2021.igem.org/Team%3ATuebingen/Description)
  + [Engineering](https://2021.igem.org/Team%3ATuebingen/Engineering)
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  + [Model](https://2021.igem.org/Team%3ATuebingen/Model)
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  + [Notebook](https://2021.igem.org/Team%3ATuebingen/Notebook)
* Human Practices
  + [Human Practices](https://2021.igem.org/Team%3ATuebingen/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3ATuebingen/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ATuebingen/Partnership)
  + [Communication](https://2021.igem.org/Team%3ATuebingen/Communication)
  + [Implementation](https://2021.igem.org/Team%3ATuebingen/Implementation)
* Team
  + [Team](https://2021.igem.org/Team%3ATuebingen/Team)
  + [Attributions](https://2021.igem.org/Team%3ATuebingen/Attributions)
* Parts
  + [Parts](https://2021.igem.org/Team%3ATuebingen/Parts)
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* [Plant Special Prize](https://2021.igem.org/Team%3ATuebingen/Plant)

Go To

[Drylab](#drylab)
[Wetlab](#wetlab)
[Download](#download)

# Experiments

Protocols of all Experiments we conducted

---

# Drylab

## Gromacs

### Installing Gromacs (Linux OS or Linux subsystem):

* Download g++ from cmake.org/download/ (cmake-version.tar.gz)
* Download fftw from fftw.org/download.html (fftw-version.tar.gz)

In terminal:

* Unpack cmake ( tar xzvf cmake-version.tar.gz)
* Move to unpacked folder (cd cmake-version)
* Run configuration (./configure)
* Install cmake (make)
* Install cmake (sudo make install)
* Unpack fftw (tar xzvf fftw-version.tar.gz)
* Move to unpacked directory (cd fftw-version)
* Run configuration (./configure)
* Install fftw (make)
* Install fftw (sudo make install)
* Install Gromacs (sudo apt-get install Gromacs)
* Success!

### Creating a membrane (All-Atom/Coarse-grained):

Membranes were created using the tool offered by CHARMM-Gui
[6,](ref6)
[7,](ref7)
[8](ref8)
.

### Predicting protein structures:

To predict the structures of our grafted cyclotides we used AlphaFold Colab
[4](ref4)
.

### Using Gromacs

As Gromacs or Molecular Dynamics simulation, in general, is a very complex and versatile topic, we are limiting the following section to what we learned about using Gromacs so far and how to set up a very basic system.

When starting with Molecular Dynamics, one should always consider that most simulations are very computationally intensive.

When the goal is to simulate a complex system (e.g., more than a protein in water) and a supercomputer is unavailable, one should consider using a so-called coarse-grained forcefield.

Forcefield is a data file used by Gromacs which contain numeric values required to compute interaction forces between different kind of atoms.

During our project we used 2 different kinds of forcefields to run our simulations:

* All-Atom: a forcefield that computes interaction forced between each atom available in the simulation, very computationally intensive.
* Coarse-grained: a forcefield that sums up groups of atoms into a single entity, describing it with the average chemical properties of the single atoms constituting it.

Thanks to coarse-grained forcefields (in our case Martini
[5](ref5)
), we were able to run some simulations on our computers as well and achieved some good results, although, as might be clear already, the resulting simulations aren't as realistic as when using an all-atom forcefield.

#### File types:

To perform any kind of simulation, Gromacs uses the so-called .gro file format.

A .gro file contains the structural information of, e.g., a protein, molecule, or a composition of different molecular structures, and the size of the virtual box within this composition are placed.

In order to create such box for e.g. a protein, one can download the .pdb file of the protein and convert it into a .gro file (see Gromacs command pdb2gmx).

Apart from the virtual box containing all components, Gromacs requires (lots) of settings to be made, so it knows precisely how to calculate forces between atoms.

Those settings for the simulation are stored in .mdp files and contain information like which method shall be used to keep the temperature or pressure of the .gro file at the given values or how long the simulation is supposed to be.

As mentioned before, there are many settings to be made here, which reflects how many scenarios Gromacs can be applied.

Next-up are topology files (.top), which (in some way) contain a resume of a .gro file by listing its different molecules by name and in the same order.

The last file required to run a successful simulation is the index file (.ndx), which allows the user to sum up atoms of the .gro file into groups by their molecule number.

Note that this file only needs to be provided by the user if any non-standard groups are being used in the .mdp file (e.g., for coupling purposes), else it's automatically generated by Gromacs.

#### Running a simulation:

If the files described in the section above are at hand, it's possible to run the simulation!

For this purpose, a .tpr file is being generated by Gromacs, which sums up all settings and files used for the simulation program.

Before a so-called "production" (the simulation we want) can be run, a couple of short pre-simulations have to be made to have a physically correct virtual box (aka .gro file).

If we skip those pre-simulations, our system could contain overlapping atoms, or isn't set to a certain temperature or pressure!

Therefore the first simulation to be run is a so-called minimization, which is often also referred to as "relaxing" the system: atoms or molecules which, e.g., overlap or show other forms of inappropriate geometry are slightly moved to bring the total energy of the system under a certain threshold (previously defined in the .mdp file).

After lowering the total energy to a realistic value, it's time to bring the system to our desired temperature (which consists of the total kinetic energy of the system).

This simulation step is called NVT equilibration, where NVT stands for number, volume, and temperature, three characteristic values of the system which are tried to keep constant.

Temperature is introduced to the system by raising the kinetic energy of its particles.

These kinetic energies are adapted until the temperature plateaus (around) the value defined in the .mdp file.

![GROMACS Energy Diagram](https://static.igem.org/mediawiki/2021/0/00/T--Tuebingen--experiments_drylab-1.png)

After obtaining the desired temperature, we need to introduce a realistic pressure to our system.

This simulation is referred to as NPT equilibration, where, instead of volume, the pressure is tried to keep constant (therefore, the volume of the system must be modified).

The NPT simulation is considered successful once the density remains constant (note that the pressure can still fluctuate widely, therefore density is used as a review metric).

![GROMACS Energy Diagram](https://static.igem.org/mediawiki/2021/3/33/T--Tuebingen--experiments_drylab-2.png)
![GROMACS Energy Diagram](https://static.igem.org/mediawiki/2021/f/f3/T--Tuebingen--experiments_drylab-3.png)

As mentioned before, pressure can still fluctuate wildly as instantaneous pressure is relatively meaningless and only makes sense as an average over time.

If the equilibration or minimization didn't yield the wanted result, it might be helpful to redo them, increasing their duration.

Now that our system has been successfully prepared, it's time for the production run (the simulation which gives us the result we want).

There are plenty of use cases for molecular dynamics simulations. Therefore, it is futile to describe a general approach in how a production run is supposed to be performed.

As a warning it may be said that although the minimization as well as the nvt and npt equilibration were successful, errors can still occur later on which may require extensive troubleshooting.

### References

1. E. Lindahl, B. H. (2001). GROMACS 3.0: A package for molecular simulation and trajectory analysis. Journal of Molecular Modeling, 306-317.
2. H. Bekker, H. B. (1993). Gromacs: A parallel computer for molecular dynamics simulations. Physics computing, 252-256.
3. H.J.C. Berendsen, D. v. (2001). GROMACS: A message-passing parallel molecular dynamics implementation. Computer Physics Communications, 306-317.
4. Jumper, J. a. (2021). Highly accurate protein structure prediction with AlphaFold. Nature, 583-589.
5. Marrink, S. J. (2007). The MARTINI force field: coarse grained model for biomolecular simulations. The journal of physical chemistry B, 7812-7824.
6. Pin-Chia Hsu, B. M. (2017). CHARMM-GUI Martini Maker for modeling and simulation of complex bacterial membranes with lipopolysaccharides. Journal of Computational Chemistry, 2354-2363.
7. Sunhwan Jo, T. K. (2008). CHARMM-GUI: A Web-based Graphical User Interface for CHARMM. Journal of Computational Chemistry, 1859-1865.
8. Yifei Qi†, H. I. (2015). CHARMM-GUI Martini Maker for Coarse-Grained Simulations with the Martini Force Field. Journal of Chemical Theory and Computation, 4486-4494.

# Wetlab

## Agarose gel electrophoresis

## Agarose gel electrophoresis

### Introduction

Separation of DNA fragments.

### Materials

* 1x TAE-buffer
* Agarose
* nitrile gloves
* loading dye: 6x purple loading dye from NEB
* DNA ladder: GenLadder 1kbp (250 bp-10000 bp)
* Diamond nucleic acid dye / Midori green
* agarose gel chamber
* power supply
* UV-desk

### Method

* Weigh 2.5 g Agarose in a 250 ml screw-cap laboratory bottle.
* Fill up to 250 ml with 1x TAE-buffer.
* Heat up in microwave until agarose completely dissolves, take out of microwave and stir every 30 to 60 s. Store bottle in 60°C incubator or store at RT and heat up in microwave before using.
* Pour agarose into gel tray. (If Midori green is used, add 1-2 µL when pouring agarose into the tray.) Let the gel solidify, covered with paper towel.
* Put the gel into electrophoresis chamber, fill up with 1x TAE-buffer until gel is slightly covered.
* Add 5 μl 6x loading dye to PCR-reaction, mix by pipetting.
* Apply whole 25 μl of PCR-reaction supplemented with loading dye and 5 µl of DNA-ladder.
* Run gel electrophoresis at 120 V for about 30 min (until dye front has run far enough).
* If no Midori green is used, stain gel in Diamond nucleic acid dye (Promega) according to their protocol.
* Analyze gel on UV-desk and take a picture.

## Control digest

## Control digest

### Introduction

To monitor cloning success.

### Materials

* Promega restriction enzymes: EcoRI (12 U/µl), BamHI (10 U/µl), HindIII (10 U/µl)
* Promega 10x restriction buffers: buffer H (EcoRI), buffer E (BamHI, HindIII)
* Promega acetylated BSA (10 mg/ml) as enzyme stabilizer
* autoclaved ddH
  2
  O
* DNA sample
* heat block

### Method

* In a sterile tube, assemble the following components in the order listed below:

  | Component | Volume (µl) |
  | --- | --- |
  | sterile ddH 2 O | to 20 |
  | 10x restriction buffer (Promega) | 2 |
  | acetylated BSA (Promega, 10 mg/ml) | 0.2 |
  | DNA sample | 0.2 - 1.5 µg |
  | restriction enzyme (Promega) | 0.5 |
  | total | 20 |
* Mix gently by pipetting, close the tube and centrifuge for a few seconds in a microcentrifuge. Incubate at 37 °C for 1 - 4 hours.
* Proceed with agarose gel analysis protocol

## *E. coli*  DH5α transformation

## *E. coli* DH5α transformation

### Introduction

Transformation of NEB 5-α chemically competent
*E. coli*
with cloning products; derived from
[NEB instructions](https://international.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987)
.

### Materials

* C2987I NEB 5-α competent
  *E. coli*
  cells
* DNA or cloning reaction
* SOC medium (NEB)
* LB selection plates
* heat block
* shaker/rotator

### Method

* Thaw a tube of NEB 5-α competent E. coli cells on ice. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
* Add 1 - 5 µl sample 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 min.
* Heat shock at 42 °C for 30 s.
* Place on ice for 5 min.
* Pipette 950 µl of SOC into the mixture.
* Place at 37 °C for 60 min. 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 100 µl of each dilution onto a selection plate and incubate overnight at 37 °C.

## GoldenGate BpiI (BbsI)

## GoldenGate BpiI (BbsI)

### Introduction

Cloning level I and level III vectors with BpiI (BbsI).

### Materials

* Nuclease free H2O
* T4 DNA ligase buffer (10x)
* Plasmids/fragments
* T4 DNA ligase (NEB)
* BbsI-HF (NEB)
* Thermocycler

### Method

* Set up 25 µl reaction in a PCR-tube by pipetting in the given order; pipet and store on ice:

  | Component | Amount |
  | --- | --- |
  | Nuclease free H2O | fill up to 25 µl |
  | T4 DNA ligase buffer (10x) | 2.5 µl |
  | Plasmids/fragments | 75 ng |
  | T4 DNA ligase (NEB) | 0.5 µl (200 U) |
  | BbsI-HF (NEB) | 1.5 µl (30 U) |
* Mix gently by pipetting up and down several times.
* Run the following program in a thermocycler:

  | Temperature (°C) | Time (min) | Cycles |
  | --- | --- | --- |
  | 37 | 5 | } 30x |
  | 16 | 5 |
  | 37 | 5 | 1x |
  | 60 | 5 | 1x |
* Transform 5 µl GoldenGate reaction into competent
  *E. coli*
  .

## GoldenGate BsaI

## GoldenGate BsaI

### Introduction

Cloning level II vectors with BsaI.

### Materials

* Nuclease free H2O
* T4 DNA ligase buffer (10x)
* Plasmids/fragments
* GoldenGate assembly mix (NEB)
* BbsI-HF (NEB)
* Thermocycler

### Method

* Set up 20 µl reaction in a PCR-tube by pipetting in the given order; pipet and store on ice:

  | Component | Amount |
  | --- | --- |
  | Nuclease free H2O | fill up to 20 µl |
  | T4 DNA ligase buffer (10x) | 2 µl |
  | Plasmids/fragments | 75 ng |
  | GoldenGate assembly mix (NEB) | 0.5 µl (200 U) |
  | BbsI-HF (NEB) | 1 µl (20,000 U/ml) |
* Mix gently by pipetting up and down several times.
* Run the following program in a thermocycler:

  | Temperature (°C) | Time (min) | Cycles |
  | --- | --- | --- |
  | 37 | 1 | } 30x |
  | 16 | 1 |
  | 37 | 5 | 1x |
  | 60 | 5 | 1x |
* Transform 5 µl GoldenGate reaction into competent
  *E. coli*
  .

## GoldenGate Esp3I

## GoldenGate Esp3I

### Introduction

Cloning cyclotides into level III vectors with Esp3I.

### Materials

* Nuclease free H2O
* T4 DNA ligase buffer (10x)
* Plasmids/fragments
* T4 DNA ligase (NEB)
* Esp3I (NEB)
* Thermocycler

### Method

* Set up 25 µl reaction in a PCR-tube by pipetting in the given order; pipet and store on ice:

  | Component | Amount |
  | --- | --- |
  | Nuclease free H2O | fill up to 25 µl |
  | T4 DNA ligase buffer (10x) | 2,5 µl |
  | Plasmids/fragments | 75 ng |
  | T4 DNA ligase (NEB) | 0.5 µl (200 U) |
  | Esp3I (NEB) | 1,5 µl (15 U) |
* Mix gently by pipetting up and down several times.
* Run the following program in a thermocycler:

  | Temperature (°C) | Time (min) | Cycles |
  | --- | --- | --- |
  | 37 | 5 | } 30x |
  | 16 | 5 |
  | 37 | 5 | 1x |
  | 60 | 5 | 1x |
* Transform 5 µl GoldenGate reaction into competent
  *E. coli*
  .

## Mini-/Midiprep

## Mini-/Midiprep

### Introduction

Producing and multiplying plasmid DNA (e.g., after cloning) from
*E. coli*
.

### Materials

* LB agar plates
* LB medium
* appropriate antibiotics for selection
* autoclaved glass test tubes
* preparation kit (NEB Monarch® Plasmid DNA Miniprep Kit)
* incubator
* nanodrop

### Method

Day 1:

* Transform competent E. coli cells and plate them on LB agar plates supplemented with the appropriate antibiotics.
* Incubate plates overnight at 37 °C

Day 2

* Add 5 ml of LB medium with appropriate antibiotics into sterile glass test tubes.
* Pick a colony from a plate with a sterile pipette tip or inoculation loop and stir in the 5 ml LB.
* Grow the bacteria overnight in shaker at 37 °C.

Day 3

* Grow bacteria for 8 h at 37 °C and shaking by inoculation of 50 ml LB with 50 µl preculture.
* Spin down liquid cultures and proceed according to the preparation protocol by NEB.
* Measure DNA concentration yielded from the preparation, using a nanodrop.

## Sequencing

## Sequencing

### Introduction

To sequence plasmid or other DNA by Sanger sequencing.

### Materials

* Sequencing reaction
* 1.5 ml Eppendorf tubes
* Labels

### Method

* Prepare plasmid according to protocol
* Prepare sequencing reaction in 1,5 ml Eppendorf tubes: 15 µl, containing 40 - 100 ng/µl plasmid/DNA and 4 µM primer

  | Component | Amount |
  | --- | --- |
  | Plasmid/DNA | V (600-1500 ng) |
  | 20 µM primer | 3 µl |
  | Autoclaved ddH 2 O | Fill up to 15 µl |
* Label the 1.5 ml Eppendorf tubes and send it to sequencing (Mycrosynth).

## Inhibition zone assay

## Inhibition zone assay

### Introduction

To test antimicrobial properties of samples against
*E. coli*
and
*B. subtilis*
.

### Materials

* LB-agar plates
* crude extract / purified protein solution
* LB medium
* *E. coli*
  cells
* *B. subtilis*
  cells
* glass tubes
* filter paper discs

### Method

Day 1:

* Inoculate cells in 10 ml LB media overnight (
  *E. coli*
  and
  *B. subtilis*
  ) in glass tube.

Day 2

* Dilute overnight culture 1:10.
* Inoculate new culture (OD
  600
  = 0.1) and let it grow for 3 - 4 h until a OD
  600
  = 0.8-1.0.
* Dilute culture 1:50.
* Prepare a series of dilutions of crude extract/protein solution: 1:10, 1:50, 1:50, 1:100, 1:500 and 1:1000 (200 μl each)
* Spread 200 μl of 1:50 bacteria cultures onto LB-agar plates and let it dry.
* Put small filter paper discs onto the LB-agar plates and add 20 μl of protein dilutions, each to one filter paper disc.
* Incubate plates overnight at 37 °C.
* Take photos of the petri dishes on the next day.

## 10x TAE-buffer

## 10x TAE-buffer

### Introduction

Standard protocol for making 10x TAE-buffer.

### Materials

* Tris-base (M = 121.14 g/mol)
* Glacial acetic acid (17,4 M)
* EDTA (M = 372.23 g/mol)
* ddH
  2
  O

### Method

* Dilute 48.5 g Tris-base, 11.4 ml glacial acetic acid and 3.7 g EDTA in 800 ml ddH
  2
  O.
* Fill up with ddH
  2
  O to 1 l.

## 1x Tricine running buffer

## 1x Tricine running buffer

### Introduction

Standard protocol for making 1x tricine running buffer.

### Materials

* 100 mM Tris-base (M = 121.14 g/mol)
* 100 mM Tricine (M = 179.172 g/mol)
* 0.1% (w/v) SDS

### Method

* Weigh in the appropriate amount of chemicals.
* Dissolve in ddH
  2
  O and set pH to 8.3.
* Fill up to desired volumed with ddH
  2
  O.

## Antibiotics

## Antibiotics

### Introduction

Preparation and storage of antibiotics.

### Materials

* Gentamicin
* Ampicillin
* Kanamycin
* Rifampicin
* Streptomycin
* Tetracycline

### Method

* Make solutions of 10 mg/ml gentamicin, 50 mg/ml ampicillin, 50 mg/ml kanamycin, 100 mg/ml rifampicin, 100 mg/ml streptomycin or 5 mg/ml tetracycline with ddH
  2
  O.
* Filter the antibiotic solutions with a 0.2 µM sterile filter under the hood and take 0.5 ml aliquots. Store them at - 20 °C.

## Media

## Media

### Introduction

Preparation of used media.

### Materials

* Müller-Hinton Broth (MH)
* Brain Heart Infusion Broth (BHI)
* LB broth
* LB agar plates

### Method

MH broth

* Dissolve 21 g of MH broth in 1 l ddH
  2
  O.
* Autoclave the solution at 121 °C for 15 min.

MH broth

* Dilute 10 ml of autoclaved MH broth with 40 ml of autoclaved water to obtain a 1/5 dilution.

MH broth

* Dissolve 37 g of BHI broth in 1 l ddH
  2
  O.
* Autoclave the solution at 121 °C for 15 min.

MH broth

* Dissolve 20 g of LB broth in 1 l ddH
  2
  O.
* Autoclave the solution at 121 °C for 15 min.

## TE buffer

## TE buffer

### Introduction

Standard protocol for making TE-buffer.

### Materials

* Tris-base (M = 121.14 g/mol)
* EDTA (M = 292.25 g/mol)
* ddH
  2
  O

### Method

* Dissolve 605.7 mg Tris and 14.6 mg EDTA in 400 ml ddH
  2
  O.
* Adjust the pH to 8.0 using a pH-meter
* Fill up to 500 ml total volume with ddH
  2
  O and autoclave.

## Acetone protein precipitation

## Acetone protein precipitation

### Introduction

To concentrate protein solutions, for SDS-PAGE or other methods not requiring properly folded proteins.

### Materials

* Acetone
* Centrifuge

### Method

* Cool acetone to -20 °C.
* Pipet protein sample solution into an Eppendorf tube and add four times the sample volume of acetone.
* Vortex tube and incubate for 60 min at -20°C.
* Centrifuge 10 min at 13.000-15.000 × g.
* Decant and remove supernatant carefully.
* Allow the acetone to evaporate from the uncapped tube at RT for 30 min.
* Add buffer appropriate for the downstream process and vortex thoroughly to dissolve protein pellet.

## *Agrobacterium*  transformation

## *Agrobacterium* transformation

### Introduction

Transformation of
*Agrobacterium tumefaciens*
with our 2in1 and 3in1 vectors for transient expression in tobacco.

### Materials

* *A. tumefaciens*
  strain AGL1
* Antibiotics: 100 mg/ml Rif, 100 mg/ml Strep, 5 mg/ml Tet, 25 mg/ml Kan
* Vector
* LB medium
* LB agar
* Shaker
* Incubator
* Centrifuge

### Method

* Thaw
  *Agrobacteria*
  on ice
* Add 3 µl plasmid DNA to 100 µl agrobacteria, mix by flicking the tube.
* Incubate 5 min on ice.
* Incubate 5 min in liquid nitrogen.
* Thaw in hand and incubate 5 min at 37 °C.
* Add 900 µl LB medium.
* Incubate 3 h at 28 °C.
* Centrifuge 2 min at 4000 rpm.
* Remove 850 µl supernatant, resuspend pellet in remaining liquid.
* Plate 100 µl of the suspension on LB agar plates containing 10 µg/ml Rif, 2.5 µg/ml Strep, 2.5 µg/ml Tet and 25 µg/ml Kan.
* Incubate plates 2 days at 28 °C.

## His-tag affinity purification

## His-tag affinity purification

### Introduction

How to purify His6-tagged peptide from tobacco crude extract. Protocol adapted from
[Promega](https://www.promega.de/-/media/files/resources/protocols/technical-bulletins/101/hislink-protein-purification-resin-protocol.pdf?rev=067f7d8a499c4554ae74fd301ad0b67b&sc_lang=en)
and
[Potula
*et*
al., 2007](https://link.springer.com/content/pdf/10.1007/s11248-007-9072-4.pdf)
.

### Materials

* HisLink™ Protein Purification Resin (Promega)
* Millipore filter (0.2 µm pore size)
* Syringes
* Column
* Dialysis chamber, 2 kDa MWCO (ThermoFisher Slide-A-Lyzer™ Dialysis Cassettes)
* Ultracentrifuge
* Magnetic stirrer
* Buffers for extraction, purification and dialysis (see table below)

| Buffers | Extraction | Binding | Wash | Elution | Dialysis |
| --- | --- | --- | --- | --- | --- |
| **Chemicals** | 150 mM NaCl | 150 mM NaCl | 150 mM NaCl | 150 mM NaCl | 150 mM NaCl |
| 3 mM KCl | 3 mM KCl | 3 mM KCl | 3 mM KCl | 3 mM KCl |
| 100 mM HEPES | 100 mM HEPES | 100 mM HEPES | 100 mM HEPES | 100 mM HEPES |
| 0.2-2 mM PMSF\* | 10 mM Imidazole | 20 mM imidazole | 500 mM Imidazole |  |
| 0.05% (v/v) Tween20\* |  |  |  |  |

\*add freshly

### Method

* Cut off and weight infiltrated leaves.
* Grind the leaves with liquid nitrogen using mortar and pestle in 200% per weight ice-cold extraction buffer.
* Incubate 1 h on ice before proceeding. Invert every 5-10 min.
* Centrifuge crude extract at 20.000 g for 30 min at 4 °C
* Collect aliquot (200 μl) from the pellet.
* Filter supernatant with Millipore filter, using pre-cleaned syringe.
* Collect aliquot (200 μl) from the filtrate
* Fill column with 1 ml HisLink resin and wait until resin has settled. Allow the column to drain, and equilibrate the resin with 5 column volumes (= 5 ml) of binding buffer, allowing the buffer to completely enter the resin bed.
* Gently add the cleared, filtered lysate to the resin until the lysate has completely entered the column.
* Collect aliquot from the flow-through.
* Wash unbound proteins from the resin using 5 ml of binding buffer.
* Collect aliquot from the flow-through.
* Wash unbound proteins from the resin using 10 ml of wash buffer.
* Collect aliquot from the wash.
* Once the wash buffer has completely entered the resin bed, add elution buffer and start collecting 0.5 ml fractions.

## Infiltration of tobacco using  *Agrobacterium tumefaciens*

## Infiltration of tobacco using *Agrobacterium tumefaciens*

### Introduction

Infect tobacco plants with genetically modified
*A. tumefaciens*
to produce our construct.

### Materials

* LB medium
* Antibiotics (100 mg/ml Rif, 100 mg/ml Strep, 5 mg/ml Tet, 25 mg/ml Kan)
* AS medium (see table below)
* Tobacco plants
* P19 bacteria solution
* Syringe (1 ml)
* Fluorescence microscope
* Shaker

| Chemicals | Concentration |
| --- | --- |
| MES-KOH buffer, pH 5.6 | 1 M |
| MgCl 2 | 3 M |
| Acetosyringone in DMSO | 150 M |

### Method

* Inoculate 2 ml of LB medium with corresponding antibiotics with a single
  *Agrobacterium*
  colony.
* Incubate over night at 28 °C while shaking.
* Centrifuge bacterial culture for 15 min at 4000 rpm and discard the supernatant. P19 culture is centrifuged as well.
* Resuspend the pellet in 2 ml of cold AS medium. Add AS medium until an OD
  600
  of 0.8-1.0.
* Combine 100 μl of the bacterial solution and 100 μl of P19 bacteria solution in a 2 ml Eppendorf tubes and fill up with AS medium to 2 ml.
* Incubate on ice for at least 1 h.
* Infiltrate 2 - 3 young tobacco leaves per plant with the bacterial solution.

![A leaf gets agroinfiltrated](https://static.igem.org/mediawiki/2021/5/50/T--Tuebingen--experiments_wetlab_transfection.jpg)

**Figure 1: Picture of how to perform the Agroinfiltration procedure.**

A 1 ml syringe is used to infiltrate the young tobacco plants from the underside of the leaves.

## Peptide purification according to Poon *et* al. 2018

## Peptide purification according to Poon *et* al. 2018

### Introduction

Protocol for extraction and purification of peptides from
*Nicotiana benthamiana*
adapted from Poon
*et*
al. 2018.

### Materials

* Acetonitrile
* Formic acid
* Filter circles, 110 mm, 5-13 µm
* Drying cabinet
* Mortal and pestle
* Magnetic stirrer
* Round-bottomed flask
* Rotation evaporator
* RP-HPLC MS
* MALDI-TOF MS

### Method

* Dry leaves overnight at 40 °C.
* Grind leaves to fine powder with mortar and pestle.
* Add 20 ml/g (dry weight) extraction buffer (50% acetonitrile - 1% formic acid in ddH
  2
  O).
* Incubate for 1 h at room temperature in a beaker glass on a magnetic stirrer.
* Filter with filter paper to remove insoluble extract into round-bottomed flask.
* Repeat incubation and filtering with insoluble extract with the same amount of extraction buffer.
* Dry soluble fraction in round-bottomed flask with rotation evaporator.

## Tricine SDS-PAGE

## Tricine SDS-PAGE

### Introduction

SDS-PAGE (with tricin instead of glycine) to get a clear separation of small protein bands (1 to 5 kDa peptides); gel casting according to Schägger 2006.

### Materials

* 10x anode buffer
* 10x cathode buffer
* 2x Tricine sample buffer
* Prestained Protein Ladder - Extra broad molecular weight (5 - 245 kDa) (Abcam)
* 3x Gel buffer: 3 M Tris, 1 M HCl, pH 8.45, 0.3 % (w/v) SDS
* Acrylamide AB-3 stock solution: 49.5 % T, 3 % C mixture
* 10 % APS
* TEMED
* Isopropanol
* heat block
* SDS gel chamber
* Power source
* Tray for staining the gel
* Shaker
* Scanner

| Buffer | 10x anode buffer   pH 8.9 | 10x cathode buffer   pH 8.25 | 2x Tricine sample buffer   pH 6.8 | 3x Gel buffer   pH 8.45 |
| --- | --- | --- | --- | --- |
| **Chemicals** | 1 M Tris | 1 M Tris | 200 mM Tris/HCl | 3 M Tris |
| 0.225 M HCl | 1 M Tricine | 2% (w/v) SDS | 1 M HCl |
|  | 1 % SDS | 40 % (v/v) glycerol | 0.3 % (w/v) SDS |
|  |  | 0.04 % (w/v) Coomassie brilliant Blue R-250 |  |
|  |  | 2 % (w/v) β-mercaptoethanol |  |

| 16 % Separation gel | Component | Amount for 30 ml |
| --- | --- | --- |
| **Chemicals** | AB-3 | 10 ml |
| 3x Gel buffer | 10 ml |
| ddH 2 O | 10 ml |
| 10 % APS\* | 100 μl |
| TEMED\* | 10 μl |

\*add latest

| 4 % Stacking gel | Component | Amount for 12 ml |
| --- | --- | --- |
| **Chemicals** | AB-3 | 1 ml |
| 3x gel buffer | 3 ml |
| ddH 2 O | 8 ml |
| 10 % APS\* | 90 μl |
| TEMED\* | 9 μl |

\*add latest

### Method

* Pour 16 % separation gel.
* Cover separation gel with isopropanol until it is fully polymerized.
* Pour 4 % stacking gel on top.
* Add comb and let gel polymerize.
* Mix 10 µl sample with 10 µl 2 x sample buffer by pipetting (For flowthroughs and crude extracts dilute 1:10 to prevent gel overload. For samples with known protein concentration use 15 - 20 µg protein).
* Heat samples at 95 °C for 10 min.
* Spin down samples shortly in centrifuge.
* Apply 15 µl of each sample onto the gel; apply 10 µl protein ladder.
* Run SDS-PAGE for about 100 min at 100 V.
* Stain gel for 20 min to 1 h in Coomassie blue staining solution, either PageBlue™ Protein Staining Solution (Thermo Fisher Scientific) or Der Blaue Jonas ultrafast protein stain (GRP), shaking at RT.
* Collect staining solution, wash gel in ddH
  2
  O for 10 min.
* Take gel pictures using the scanner.

## Western blot

## Western blot

### Introduction

To specifically detect proteins separated by SDS-PAGE using antibodies.

### Materials

* 1x Blotting buffer
* TBS-T
* Milk-powder
* 100 % Methanol (can be reused)
* 1x PVDF membrane (Immobilion)
* 4x Whatman-paper, 8.6 x 6.2 cm
* Mouse anti-His antibody
* Anti-mouse HRP-coupled antibody
* Luminescence reagents (Roche Lumi-Light Western Blotting substrate)
* Semi-dry blotter
* Rotator

| Buffer | 1x blotting buffer | TBS-T pH 7.4 |
| --- | --- | --- |
| **Chemicals** | 25 mM Tris | 150 mM NaCl |
| 19.2 mM glycerol | 0.1 % Tween20 |
| 20 % (v/v) MeOH | 50 mM Tris/HCl |

### Method

* Perform SDS-PAGE
* Activation of the PVDF-membrane in 100 % MeOH; afterwards equilibration in blotting buffer.
* Equilibration of Whatman-papers (4 X) and SDS-gel in blotting buffer.
* Assemble blotting sandwich:
![Assemble blotting sandwich](https://static.igem.org/mediawiki/2021/c/c7/T--Tuebingen--experiments_wetlab_westernblot.jpg)

**Figure 2: Set-up of the blotting sandwich for western blotting.**
We used a Semi-Dry blotting technique. Therefore, the PVDF membrane was activated in methanol and two layers of Whatman paper, preequilibrated in blotting buffer, were placed above and under gel or membrane, respectively.

* Run blot at 20 V (1000 mA) for 30 min.
* Block the membrane for 1-2 h at RT (
  or
  O.N. at 4 °C) on a rotator with 5% milk-powder/TBS-T
* Wash 3 times (each 10 - 15 min) with TBS-T.
* Transfer membrane in a clean 50 ml tube.
* Incubate with mouse anti-His antibody (diluted 1:1000 (10 µl AB in 10 ml TBS-T or 5% milk/TBS-T) rotating O.N. at 4 °C (
  or
  for 1 h rotating at RT).
* Wash 3 times (each 10 - 15 min) with TBS-T.
* Incubate with anti-mouse HRP-coupled antibody (diluted 1:5000 (2 µl in 10 ml TBS-T); wrap the 50 ml tube in aluminum foil to protect the HRP-coupled antibody from light damage) for 1 h rotating at RT.
* Wash 3 times (each 10 - 15 min) with TBS-T.
* Mix detecting reagent A and B in a 1:1 ratio (300 µl A and 300 µl B, for 6.2 x 8.6 cm membrane). Mix the reagents shortly before use in an Eppendorf tube wrapped in aluminum foil and store it on ice.
* Put the membrane on a plastic foil, distribute detection reagent on the membrane, cover the membrane with another plastic foil and remove air bubbles. Incubate for 5 min at room temperature (covered from light).
* Detect luminescence with increasing exposure time step-wise (e. g. 5 s, 1 min, 5 min, 10 min). Take a picture of the ladder with an exposure time of 0.1 s.

## Activity Testing – microdilution assay (Koehbach  *et*  al. 2021 / Wiegand  *et*  al. 2008)

## Activity Testing – microdilution assay (Koehbach *et* al. 2021 / Wiegand *et* al. 2008)

### Introduction

Purified construct or crude extract will be used to test the antimicrobial activity. We will use several different methods for testing since the antimicrobial effects of AMPs can be hidden by different factors of an assay. To overcome this problem, we will do many different assays.

### Materials

* 96-well plates (sterile, polypropylene, U-bottom (Nunclon Delta polystyrene))
* Mueller-Hinton I Broth (MH)
* Brain Heart Infusion Broth (BHI)
* *E. coli*
  K12
* *Bacillus subtilis*

### Method

**Assay with MH Broth**

* Twofold serial dilution of the antibiotics and peptides or of crude extracts using the respective media (MH, 1/5 MH, BHI).
* For sterility control use media without bacterial solution.
* For growth control use media with bacteria but without peptide/antibiotics.
* Prepare 10 ml of a 1x10
  6
  CFU/ml (bacterial cultures should be in log-phase):

  For this, take 3 - 5 colonies of a plate and resuspend in 500 µl saline (0.9 % NaCl).

  Pipette 100 µl of this bacterial suspension into a cuvette and add 900 µl saline (1:10 dilution). Measure OD
  600
  for CFU/ml determination.

  Dilute cells depending on needed concentration (OD
  600
  = 0.1 equals
  *E. coli*
  BW25113: 1.4x10
  8
  CFU/ml;
  *B. subtilis*
  168: 2x10
  7
  CFU/ml.)
* Add 50µL bacteria suspension with the needed concentration to the wells.
* Incubate plates at 37 °C O.N.
* Measure OD
  600
  .

## SDS-extraction

## SDS-extraction

### Introduction

We could not extract our peptide with the former extraction buffer (100 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM KCl), but with Lämmli buffer containing SDS. Therefore, extraction might be successful with a new extraction buffer containing SDS.

### Materials

* extraction buffer
* precipitation buffer
* Mortar and pestle
* centrifuge
* dialysis chamber
* exicator

| Buffer | extraction buffer pH 8.8 | Precipitation buffer |
| --- | --- | --- |
| **Chemicals** | 125 mM Tris/HCl | 90 % (v/v) acetone |
| 10 % (v/v) glycerol | 5 % (v/v) triethylamine |
| 1 % (w/v) SDS | 5 % (v/v) acetic acid |

### Method

* Cut off and weight infiltrated leaves.
* Grind the leaves with liquid nitrogen using mortar and pestle.
* Add 200 % (v/w) extraction buffer
* Centrifuge shortly to move down plant remnants, mix briefly afterwards.
* Incubate on ice for 1 h.
* Centrifuge 2 x 10 min with 4200 rpm, at 4 °C.
* Take supernatant into dialysis chamber, dialysis at 4 °C O.N.
* Take sample out of the dialysis chambers.
* Add 19 x volume of precipitation buffer, mix gently, incubate on ice for 1 h.
* Centrifuge 10 min at 4 °C with 4200 rpm, discard supernatant.
* Wash pellet 2 x with 1 ml precipitation buffer.
* Wash pellet 2 x with 1 ml acetone.
* Dry pellet in the exicator.
* Dissolve pellet in destination buffer.

## Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels

## Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels

### Introduction

Protein bands are excised from SDS-PAGE gels, destained, and extracted for subsequent mass spectrometric analysis of whole proteins.

### Materials

* Acetonitrile
* Ammonium bicarbonate
* Trifluoroacetic acid
* HEPES extraction buffer for re-solubilisation (10 mM HEPES, 15 mM NaCl, 0.3 mM KCl, pH 7.5)

| Buffer | destaining buffer | extraction buffer |
| --- | --- | --- |
| **Chemicals** | 50 % (v/v) acetonitrile | 50 % (v/v) acetonitrile |
| 50 % (v/v) ddH20 | 45 % (v/v) ddH20 |
| (+ 100 mM (NH4)HCO3) | 5 % (v/v) TFA |

### Method

* The respective protein bands were excised from the Coomassie-stained SDS-PAGE gels with a scalpel and transferred into 1.5 ml tubes.
* Destaining of the gel slices was performed two times in 1 ml destaining buffer 1 (AcN:ddH
  2
  O 1:1) and two times in 1 ml destaining buffer 2 (buffer 1 + 100 mM (NH
  4
  )HCO
  3
  ). The tubes were placed on a heat block (40 °C) for about 15 minutes and rigorously vortexed in between until the blue color faded.
* Afterwards the buffer was removed and the gel slices were crushed using tweezers. The crushed gel slices were then incubated in 1 ml extraction buffer at 30 °C for about two hours on a rotator.
* Thereafter the solvent was evaporated on a vacuum concentrator O.N. and the extract was re-solubilized in 10 µl of 1:10 diluted HEPES extraction buffer.
* After the first measurements the samples were additionally concentrated and desalted using C18 resin.
* The samples were analyzed via MALDI-TOF MS (Bruker) and LC-MS.

# Download

[Drylab Experiments as pdf (Download)](https://static.igem.org/mediawiki/2021/b/b5/T--Tuebingen--experiments_drylab.pdf)

[Wetlab Experiments as pdf (Download)](https://static.igem.org/mediawiki/2021/6/66/T--Tuebingen--experiments_wetlab.pdf)

### About Us

We are the iGEM Team Tuebingen, a group of motivated students who are working on creating a fast screening platform for stabilized peptides. We are aiming to provide a system that gives everyone the ability to stabilize peptides such as antimicrobial peptides to create better medical agents.

### Follow Us

### Contact Us

#### team@igem-tuebingen.de

#### +49 7071 9189 745

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# Team:Tuebingen/Results

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# Results

On the following pages we present to you the results of our various experiments.

---

* [Overview](#main_results)
* [Cloning](#cloning)
* [Expression in
  *Nicotiana*](#expression)
* [Extraction and Purification](#extraction)
* [Antimicrobial Assays](#assays)
* [Model](#model)

Go To

[Overview](#res_mai_main)
[Final Vectors](#res_mai_fina)

# Overview

For a better overview and easier understanding of our results, we structured the results part into the four main experimental steps
*Cloning*
,
*Expression in
*Nicotiana**
,
*Extraction and Purification*
, and
*Antimicrobial Assays*
. This overview page provides the summarized most important results for the seven different constructs we worked on. More details and discussion for these results are found on the other result pages.

On the
Cloning
page, we explain our cloning schedule, our cloning achievements in the lab, and what they mean for our overall project. In the next step, we explain our plant expression system and how it performed in the lab on the
Expression in
*Nicotiana*
page. On the
Extraction and Purification
page, we then discuss our attempts to purify our cyclotides from the plants. The final essential experimental step in our project is the antimicrobial activity testing of our extracted and purified cyclotides. Our assays and their performance are found on the
Antimicrobial Assays
page.

## Final Vectors

If you want to get an overview of all important information at one glance, you can easily click on the plasmids in our cloning scheme to find out which experiments we conducted with it and what the respective results are.

### 2in1 Vectors

### 3in1 Vectors

![2in1 CHEN_1 Vector](https://static.igem.org/mediawiki/2021/c/c9/T--Tuebingen--results_cloning_2in1_CHEN_1.png)

##### 2in1 CHEN\_1 Vector

#### Cloning

Green fluorescent protein (GFP), precursor and cyclotide with CHEN grafted in the first loop and a His-tag in the fifth loop were successfully cloned into our plasmid.

#### Transient Expression in *N. benthamiana*

Green fluorescence from infiltrated
*N. benthamiana*
leaves could be observed, which shows successful infiltration and transient expression of genes from this vector.

#### Extraction and Purification

We aimed to extract the cyclotide from the infiltrated tobacco leaves with an SDS-based buffer (2% SDS + 5% β-mercaptoethanol). The success of the extraction could not be confirmed by neither a specific band on the Coomassie-stained tricine SDS-PAGE gel, nor a specific band in the anti-His western blot.

We did not perform any purification experiments for this construct.

![Fluorescence of GFP in tobacco leaf, 2in1-CHEN_1](https://static.igem.org/mediawiki/2021/e/e2/T--Tuebingen--results_mainpage_2in1_CHEN_1.jpg)

*Figure 1:*
Green fluorescence showing GFP expression in a tobacco leaf transfected with our 2in1\_c\_CHEN\_1 construct by agroinfiltration observed by fluorescence microscopy.

#### Antimicrobial activity

We did not perform any antimicrobial activity testing experiments for this construct.

![cloning schedule](https://static.igem.org/mediawiki/2021/a/a0/T--Tuebingen--results_cloning_3in1_CHEN_1.png)

##### 3in1 CHEN\_1 Vector

#### Cloning

Green fluorescent protein (GFP), asparagine endopeptidase 1 from
*Clitoria ternatea*
(CtAEP1), precursor and cyclotide with CHEN grafted in the first loop and a His-tag in the fifth loop were successfully cloned into our plasmid.

#### Extraction and Purification

We aimed to extract the cyclotide from the infiltrated tobacco leaves with various buffer compositions, for instance different HEPES buffers (with or without 0.05% Tween20) and SDS-based buffers (1% SDS and 2% SDS + 5% β-mercaptoethanol). The success of the extraction could not be confirmed in any of the buffers by neither a specific band on the Coomassie-stained tricine SDS-PAGE gel, nor a specific band in the anti-His western blot.

To get a purified cyclotide, we aimed to perform His-tag purification using Ni-NTA resin. The success of the purification couldn’t be confirmed by neither a specific band on the Coomassie-stained tricine SDS-gel, nor a specific band in the anti-His western blot, nor a detectable protein concentration in the eluate after purification.

#### Transient Expression in *N. benthamiana*

Green fluorescence from infiltrated N. benthamiana leaves could be observed, which shows successful infiltration and transient expression of genes from this vector.

![Fluorescence of GFP in tobacco leaf, 3in1-CHEN_1](https://static.igem.org/mediawiki/2021/8/88/T--Tuebingen--results_mainpage_3in1_CHEN_1.jpg)

*Figure 1:*
Green fluorescence showing GFP expression in a tobacco leaf transfected with our 3in1\_c\_CHEN\_1 construct by agroinfiltration observed by fluorescence microscopy.

#### Antimicrobial activity

Neither the crude extract, nor the potentially purified cyclotide showed any antimicrobial activity in neither the inhibition zone assay, nor the microdilution assay.

![cloning schedule](https://static.igem.org/mediawiki/2021/9/98/T--Tuebingen--results_cloning_3in1_KR-12_1.png)

##### 3in1 KR-12\_1 Vector

#### Cloning

Green fluorescent protein (GFP), asparagine endopeptidase 1 from
*Clitoria ternatea*
(CtAEP1), precursor and cyclotide with KR-12 grafted in the first loop and a His-tag in the fifth loop were successfully cloned into our plasmid.

#### Transient Expression in *N. benthamiana*

Green fluorescence from infiltrated
*N. benthamiana*
leaves could be observed, which shows successful infiltration and transient expression of genes from this vector.

#### Extraction and Purification

We aimed to extract the cyclotide from the infiltrated tobacco leaves with an SDS-based buffer (1% SDS). The success of the extraction could not be confirmed by neither a specific band on the Coomassie-stained tricine SDS-PAGE gel, nor a specific band in the anti-His western blot.

We did not perform any purification experiments for this construct.

![Fluorescence of GFP in tobacco leaf, 3in1-KR-12_1](https://static.igem.org/mediawiki/2021/e/e8/T--Tuebingen--results_mainpage_3in1_KR-12_1.jpg)

*Figure 1:*
Green fluorescence showing GFP expression in a tobacco leaf transfected with our 3in1\_c\_KR-12\_1 construct by agroinfiltration observed by fluorescence microscopy.

#### Antimicrobial activity

We did not perform any antimicrobial activity testing experiments for this construct.

![cloning schedule](https://static.igem.org/mediawiki/2021/9/90/T--Tuebingen--results_cloning_2in1_CHEN_6.png)

##### 2in1 CHEN\_6 Vector

#### Cloning

Green fluorescent protein (GFP), precursor and cyclotide with CHEN grafted in the sixth loop and a His-tag in the fifth loop were successfully cloned into our plasmid.

#### Transient Expression in *N. benthamiana*

Green fluorescence from infiltrated
*N. benthamiana*
leaves could be observed, which shows successful infiltration and transient expression of genes from this vector.

#### Extraction and Purification

We aimed to extract the cyclotide from the infiltrated tobacco leaves with various buffer compositions, for instance an acidic acetonitrile-based buffer and an SDS-based buffer (2% SDS + 5% β-mercaptoethanol). The success of the extraction could not be confirmed in any of the buffers by neither a specific band on the Coomassie-stained tricine SDS-PAGE gel, nor a specific band in the anti-His western blot.

We did not perform any purification experiments for this construct.

![Fluorescence of GFP in tobacco leaf, 2in1-CHEN_6](https://static.igem.org/mediawiki/2021/0/0f/T--Tuebingen--results_mainpage_2in1_CHEN_6.jpg)

*Figure 1:*
Green fluorescence showing GFP expression in a tobacco leaf transfected with our 2in1\_c\_CHEN\_6 construct by agroinfiltration observed by fluorescence microscopy.

#### Antimicrobial activity

We did not perform any antimicrobial activity testing experiments for this construct.

![cloning schedule](https://static.igem.org/mediawiki/2021/3/38/T--Tuebingen--results_cloning_3in1_CHEN_6.png)

##### 3in1 CHEN\_6 Vector

#### Cloning

Green fluorescent protein (GFP), asparagine endopeptidase 1 from
*Clitoria ternatea*
(CtAEP1), precursor and cyclotide with CHEN grafted in the sixth loop and a His-tag in the fifth loop were successfully cloned into our plasmid.

#### Extraction and Purification

We aimed to extract the cyclotide from the infiltrated tobacco leaves with various buffer compositions, for instance a HEPES buffer (without 0.05% Tween20), an acidic acetonitrile-based buffer and SDS-based buffers (1% SDS and 2% SDS + 5% β-mercaptoethanol). The success of the extraction could not be confirmed in any of the buffers by neither a specific band on the Coomassie-stained tricine SDS-PAGE gel, nor a specific band in the anti-His western blot.

To get a purified cyclotide, we aimed to perform His-tag purification using Ni-NTA resin. The success of the purification couldn’t be confirmed by neither a specific band on the Coomassie-stained tricine SDS-gel, nor a specific band in the anti-His western blot, nor a detectable protein concentration in the eluate after purification.

#### Transient Expression in *N. benthamiana*

Green fluorescence from infiltrated
*N. benthamiana*
leaves could be observed, which shows successful infiltration and transient expression of genes from this vector.

![Fluorescence of GFP in tobacco leaf, 3in1-CHEN_6](https://static.igem.org/mediawiki/2021/f/f5/T--Tuebingen--results_mainpage_3in1_CHEN_6.jpg)

*Figure 1:*
Green fluorescence showing GFP expression in a tobacco leaf transfected with our 3in1\_c\_CHEN\_6 construct by agroinfiltration observed by fluorescence microscopy.

#### Antimicrobial activity

Neither the crude extract, nor the potentially purified cyclotide showed any antimicrobial activity in neither the inhibition zone assay, nor the microdilution assay.

![cloning schedule](https://static.igem.org/mediawiki/2021/c/ce/T--Tuebingen--results_cloning_3in1_KR-12_6.png)

##### 3in1 KR-12\_6 Vector

#### Cloning

Green fluorescent protein (GFP), asparagine endopeptidase 1 from
*Clitoria ternatea*
(CtAEP1), precursor and cyclotide with KR-12 grafted in the first loop and a His-tag in the fifth loop were successfully cloned into our plasmid.

#### Transient Expression in *N. benthamiana*

Green fluorescence from infiltrated
*N. benthamiana*
leaves could not be observed.

#### Extraction and Purification

We did not perform any purification experiments for this construct.

#### Antimicrobial activity

We did not perform any antimicrobial activity testing experiments for this construct.

![cloning schedule](https://static.igem.org/mediawiki/2021/6/6b/T--Tuebingen--results_cloning_3in1_empty.png)

##### 3in1 empty Vector

#### Cloning

Green fluorescent protein (GFP), precursor and cyclotide with CHEN grafted in the first loop and a His-tag in the fifth loop were successfully cloned into our plasmid.

#### Extraction and Purification

We aimed to extract the cyclotide from the infiltrated tobacco leaves with various buffer compositions, for instance different HEPES buffers (with or without 0.05% Tween20) and SDS-based buffers (1% SDS and 2% SDS + 5% β-mercaptoethanol). The success of the extraction could not be confirmed in any of the buffers by neither a specific band on the Coomassie-stained tricine SDS-PAGE gel, nor a specific band in the anti-His western blot.

To get a purified cyclotide, we aimed to perform His-tag purification using Ni-NTA resin. The success of the purification couldn’t be confirmed by neither a specific band on the Coomassie-stained tricine SDS-gel, nor a specific band in the anti-His western blot, nor a detectable protein concentration in the eluate after purification.

#### Transient Expression in *N. benthamiana*

Green fluorescence from infiltrated N. benthamiana leaves could be observed, which shows successful infiltration and transient expression of genes from this vector.

![Fluorescence of GFP in tobacco leaf, 3in1-c_blank](https://static.igem.org/mediawiki/2021/2/26/T--Tuebingen--results_mainpage_3in1_c_blank.jpg)

*Figure 1:*
Green fluorescence showing GFP expression in a tobacco leaf transfected with our 3in1\_c\_blank construct by agroinfiltration observed by fluorescence microscopy.

#### Antimicrobial activity

Neither the crude extract, nor the potentially purified cyclotide showed any antimicrobial activity in neither the inhibition zone assay, nor the microdilution assay.

Go To

[Our Cloning Levels](#res_clo_ourc)
[Why we used Golden Gate Cloning](#res_clo_whyw)
[Testing our System](#res_clo_test)
[The Efficiency of our System](#res_clo_effi)
[References](#res_clo_refe)

# Cloning

* *We designed a cloning schedule leading to a vector that enabled us to express GFP, the asparagine endopeptidase, and a cyclotide with a grafted AMP from one vector in plants.*
* *We successfully tested the system, cloned our constructs, and proved the high efficiency of our system.*

## Our cloning Levels

We used four levels of Golden Gate cloning to assemble our final vectors, including green fluorescent protein (GFP), the asparagine endopeptidase (AEP), and the cyclotide precursor with the different grafted cyclotide constructs. In the first cloning level, we cloned our 35S promoter, 35S terminator, the precursor coding sequence, our antimicrobial peptide (AMP)-containing cyclotide, or the tag (HA, myc) into a LI backbone. In the second level, we assembled single genes into one vector by combining promoter, coding sequence, terminator, and tag sequences from the first level. In the third level, we now combined these whole genes into one vector.

![cloning schedule](https://static.igem.org/mediawiki/2021/e/e4/T--Tuebingen--results_cloning_schedule.png)

###### Cloning Schedule

[GFP](parts.html?trgt=BBa_K3669012)

[precursor](parts.html?trgt=BBa_K3757001)

[CtAEP1](parts.html?trgt=BBa_K3757000)

*lacZ*

[cyclotide](parts.html?trgt=parts_collection)

[P 35S](parts.html?trgt=BBa_K788000)

[T 35S](parts.html?trgt=BBa_K1159307)

[HA tag](parts.html?trgt=BBa_K3183024)

[c-myc tag](parts.html?trgt=BBa_K3757008)

[His tag](parts.html?trgt=BBa_M50428)

[LB/RB](parts.html?trgt=BBa_K233326)

dummy

We built two kinds of level III vectors: 3in1 vectors containing the cyclotide precursor, the AEP and GFP, and 2in1 vectors containing only the precursor and GFP as a negative control. We called the vectors from this level “empty”, as they didn't contain our AMP yet. In the last level, we now cloned our AMP into the precursor in the empty LIII vectors. We labelled these vectors according to the grafted AMP and its position. So, c\_CHEN\_1 indicates that the cyclotide contains the CHEN AMP in loop number 1. We called the negative control cyclotide without any grafted AMP c\_blank. Furthermore, all used cyclotides contained a His-Tag in loop 5.

## Why we used Golden Gate Cloning

We chose a Golden Gate based cloning method as this system not only allowed us to assemble three genes into one single vector, but also to very quickly exchange the AMPs in the cyclotide precursor for grafting once the empty level III vector is assembled. These are the most important requirements our final vector had to fulfill. We needed to express three genes at the same time: our AMP grafted into the cyclotide precursor, the AEP to convert the precursor into a cyclotide, and GFP as an expression control. In theory, these genes could also be placed on different vectors and plants could be co-infiltrated with the different systems.

However, we highly preferred this 3in1 (3 genes in 1 vector) system, as this system ensures that AEP and cyclotide are expressed in the same cells, and all cells showing green fluorescence should also express the other two genes. Therefore, we regarded this system to be less error-prone and easier to handle. Building this vector by Golden Gate cloning was our preferred method, as we had access to basic vectors for this system including terminators, promoters, and tags. These vectors were kindly provided by the research group of Prof. Dr. Klaus Harter from the plant physiology at the ZMBP.

## Testing our System

The vectors were assembled by Golden Gate reactions, transformed into
*Escherichia coli*
, and plasmids were purified from overnight cultures by Miniprep. All our cloning steps were successful, as confirmed by control digest and sequencing. The results produced for these steps are shown for the example of the c\_blank vector in figure 1.

![results of our Golden Gate reaction](https://static.igem.org/mediawiki/2021/e/ec/T--Tuebingen--results_cloning_Fig1_quer.jpg)

*Figure 1: Confirming the success of our Golden Gate reaction.*
(A) Colony growth. Only correct colonies are able to grow under the selective antibiotic pressure of kanamycin. If any level I vector would be present after the reaction, bacteria transformed with this vector wouldn't be able to grow on this plate. Non-reacted level III vectors would be able to grow, but can be recognized by blue-white screening, as shown in figure 2. The area in the red rectangle is shown in figure 2 in more detail. (B) Restriction digest. Digesting the by Golden Gate cloning assembled vector with HindIII leads to a different pattern compared to the empty vector before the Golden Gate reaction, as the new cyclotide insert replaces a part in the precursor that contained an additional HindIII restriction site. The restriction digest shows that all three colonies contain the insert. (C) Sequencing. To confirm the correct sequence of the synthesized insert, we sent our purified vectors of level I for sequencing. Aligning the
sequences to our virtually cloned vectors in SnapGene shows that the sequence of our clone is correct. Sequencing was done with a forward (fw) and a reverse (rev) primer, red bars show correctly aligned base pairs, and white parts mismatches.

The correct cloning of the cyclotide with the respective AMP into our empty LIII 3in1 vector could also be verified through blue-white screening of the grown colonies on LB-agar plates supplemented with X-Gal. The empty LIII 3in1 vector contains a
*lac-*
operon, which consists of the
*lac*
promoter,
*lac*
operator,
*lacZ*
and
*lacI*
gene, at the cloning site, where the gene sequence coding for the precursor of the cyclotide (LI constructs c\_blank, c\_CHEN, or c\_KR-12) was inserted by Esp3I restriction enzymes. The
*lac*
promoter is inducible, therefore transcription of the genes under its control only takes place when lactose or a lactose analog like Isopropyl-β-D-thiogalactopyranoside (IPTG) is added to the growth medium as it is otherwise repressed by an repressor encoded by the
*lacI*
gene.

The
*lacZ*
gene encodes for the enzyme β-galactosidase, which hydrolyses X-Gal to galactose and a substance, that turns blue upon oxidation
[1](#res_clo_juli)
. Hence, we could recognize successfully cloned colonies by their color because bacteria carrying the LIII 3in1 plasmid with the cyclotide precursor appeared white and not blue due to them not expressing β-galactosidase.

![blue-white-screening](https://static.igem.org/mediawiki/2021/6/64/T--Tuebingen--results_cloning_Fig2.jpg)

*Figure 2: Blue-white screening.*
Blue-white screening enabled us to distinguish between empty level III (blue colonies) and level IV vectors (white colonies). The fact, that there are far more white than blue colonies showed us, that our Golden Gate assembly protocol is very efficient.

## The Efficiency of our System

In the end, our established cloning system enabled us to clone different grafted AMPs in a fast and efficient way. All our cloning steps directly worked, proving the high efficiency of our protocols. After the initial cloning of the level III vectors, new AMP constructs can be cloned into the final vector within 5 days. Therefore, the new AMPs have to be synthesized, which is cost-efficient due to their small size. To clone the synthesized fragments into the level III vectors within 5 days, the following schedule should be used:

| Day | Step |
| --- | --- |
| Day 1 | Golden Gate reaction level one (synthesised fragment into level I vector), transformation of competent *E.coli* cells |
| Day 2 | Pick colonies and set overnight cultures for Miniprep for plasmid purification |
| Day 3 | Miniprep, confirm correct clones by control restriction digest, Golden Gate reaction level IV, transformation of competent *E.coli* cells |
| Day 4 | Pick colonies (blue/white screening) and set overnight cultures for Miniprep for plasmid purification |
| Day 5 | Miniprep, confirm correct clones by control restriction digest and we have our purified plasmid for Agrobacterium transformation |

This Scheme shows the different steps of our established cloning system.

# References

1
Julin, D. A. (2018). Blue/White Selection. In R. D. Wells, J. S. Bond, J. Klinman, & B. S. S. Masters (Eds.), Molecular Life Sciences: An Encyclopedic Reference (pp. 72–73). Springer New York. https://doi.org/10.1007/978-1-4614-1531-2\_94

Go To

[Procedure](#res_exp_proc)
[Transformation](#res_exp_tran)
[Infiltration of Tobacco Leaves](#res_exp_infi)
[Fluorescence Microscopy](#res_exp_fluo)
[Discussion](#res_exp_disc)
[References](#res_exp_refe)

# Expression in *Nicotiana*

* *All final vectors were successfully transformed in
  *Agrobacteria*
  , as confirmed by colony growth on the respective selective plates.*
* *Successful transfection of plants and expression of our control was observed via fluorescence for all except one construct.*

## Procedure

After successful cloning, the purified vectors were transformed into
*Agrobacterium tumefaciens*
for the following agroinfiltration of tobacco plants. After growing the plants for 4 days, the recombinant protein expression of our control green fluorescent protein (GFP) was confirmed by fluorescence microscopy and the leaves were harvested for protein extraction and purification.

## Transformation of *Agrobacterium tumefaciens*

The successful transformation of
*A. tumefaciens*
was confirmed by colony growth on the respective selective plates. We used the strain AGL1 which grows relatively slow, but has a high transfection rate and expression level. We successfully transformed all our level four vectors including our two negative controls for cyclisation with 2in1 vectors without the AEP gene, and 3in1 constructs containing the empty cyclotide precursor as a control (c\_blank), grafted CHEN constructs (c\_CHEN\_1 and c\_CHEN\_6), and grafted KR12 constructs (c\_KR-12\_1 and c\_KR-12\_6).

![colony growth of agrobacterium tumefaciens](https://static.igem.org/mediawiki/2021/0/00/T--Tuebingen--results_agros_3in1_16.jpg)

*Figure 1: Colony growth of Agrobacterium tumefaciens transformed with the 3in1 c\_CHEN\_6 construct on the respective selective plate.*
A bacterial suspension of
*A. tumefaciens*
strain AGL1 was transformed according to protocol and afterwards plated on LB agar plates containing 10 µg/ml rifampicin, 2.5 µg/ml streptomycin, 2.5 µg/ml tetracycline and 25 µg/ml kanamycin. The plates were incubated at 28 °C for two days before inoculation of an O.N. culture with the grown colonies for later infiltration of tobacco.

## Infiltration of Tobacco Leaves

*Nicotiana benthamiana*
leaves were infiltrated with
*A. tumefaciens*
containing the respective vectors and a p19 suppressor of gene-silencing construct. Co-transfection of p19 is essential, as p19 prevents that the addition of the vector leads to RNA-induced gene silencing
[1](#res_exp_voin)
. We infiltrated the tobacco leaves by syringe infiltration for transient protein expression. As a negative control, leaves were infiltrated with
*Agrobacteria*
containing only the p19 construct.

## Fluorescence Microscopy

![fluorescence microscopy images of infiltrated leaves](https://static.igem.org/mediawiki/2021/9/91/T--Tuebingen--results_expression_fluoro_figure2.jpg)

*Figure 2: Microscopy images from untransfected N. benthamiana leaves and leaves infiltrated with the respective 2in1 and 3in1 vectors.*
GFP – fluorescence channel, BF – brightfield microscopy, scale bar – 50.0 µm

After growing the plants for 4 days, the expression of our expression control protein GFP was confirmed by fluorescence microscopy. In comparison to the negative control, leaves transfected with our final vectors showed clear green fluorescence for all except the c\_KR-12\_6 construct (figure 2). As a negative control, non-infiltrated leaves or leaves infiltrated with p19 only were checked for green fluorescence. As shown in figure 2, this negative control showed no significant level of green fluorescence, confirming that the green fluorescence we detected for other vectors can only result from successful expression of one of our final vectors.

## Discussion

We can draw the conclusion that the transfection was successful for both the 2in1 and the 3in1 vectors. As the sequences coding for our antimicrobial peptide (AMP) constructs are located on the same vector as the sequence encoding for the GFP, the AMPs should be expressed in the plant cells, as further discussed in the
Extraction and Purification
section. For the c\_KR-12\_6 construct, where no fluorescence was observed, infiltration was only tried once and the failure could be explained by procedural inaccuracies like insufficient resuspension of the
*Agrobacteria*
-containing infiltration medium, leading to infiltration with a too low
*Agrobacteria*
concentration. Concluding from the fluorescence microscopy results, we can summarize that our transfection strategy is efficient and works for different AMP constructs.

However, not the whole infiltrated leaves were fluorescent but defined foci only, which indicates a low expression rate. As a further attempt to analyze the effectiveness of the agroinfiltration, GFP expression in the whole leave and also the whole plant could be analyzed under UV light.

Furthermore, to increase the efficiency of agrotransfection and especially the expression of the desired genes, viral vectors could be used instead of a non-replicating construct for the transient expression. RNA viral-based vectors combine several advantages in one, such as the ability of the replicons to move into neighboring plant cells by cell-to-cell movement, and therefore promise high expression yields of recombinant proteins in plants. The successful
*Agrobacterium*
-mediated delivery of a tobacco mosaic virus (TMV)-based vector into
*N. benthamiana*
leaves has already been shown
[2](#res_exp_mari)
and it would therefore be a reasonable continuation of our project to attempt to reach higher expression yields of our cyclotides with a different vector.

# References

1
Voinnet, O., Rivas, S., Mestre, P., & Baulcombe, D. (2003). An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. The Plant Journal: For Cell and Molecular Biology, 33, 949–956. https://doi.org/10.1046/j.1365-313X.2003.01676.x

2
Marillonnet, Sylvestre; Thoeringer, Carola; Kandzia, Romy; Klimyuk, Victor; Gleba, Yuri (2005): Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. In: Nature biotechnology 23 (6), S. 718–723. DOI: 10.1038/nbt1094.

Go To

[First Attempts](#res_ext_abaa)
[Troubleshooting](#res_ext_acaa)
[Further Experiments](#res_ext_adaa)
[MADLI-TOF MS](#res_ext_adda)
[References](#res_ext_refe)

# Extraction and Purification

* *Our standard protocols for extraction and purification did not work for our cyclotides.*
* *Troubleshooting and further experiments allowed us to exclude some potential problems that would explain the protocol failure and we assumed the buffer to be the most likely problem.*
* *MALDI-TOF MS was able to confirm the presence of peptides with masses highly similar to the cyclic constructs c\_CHEN\_1 and c\_CHEN\_6 in our extracts.*

## First Attempts of Extraction and Purification of our Cyclotides

The harvested leaves were frozen in liquid nitrogen and stored at - 20 °C before being pestled for extraction. The extraction was performed in HEPES buffer, whose composition was adjusted to the recommendations for the following His-purification via HisLink Protein Purification Resin (commercially available at Promega)
[1](#res_ext_prom)
. Apart from the buffer we followed a protocol, which was shown to be suitable for the purification of plant peptides
[2](#res_ext_potu)
.

The steps of the purification were analyzed on an SDS-PAGE gel and by western blotting using an anti-His antibody. We used tricine-SDS gels that are suitable for the separation of small peptides
[3](#res_ext_scha)
. The first round of extraction and following purification was done with around 2 g of plant leaf tissue and a ratio of 1:2 plant mass:extraction buffer volume. In the extract, a total protein concentration between 0.6 to 1 mg/ml was detected. Afterwards purification via Ni-NTA was performed and the eluted fractions were then pooled and dialysed against HEPES buffer O.N. to remove the imidazole of the elution buffer. In this first experiment, no remarkable band was found in neither the Coomassie-stained SDS-PAGE gel, nor the western blot. Furthermore, the protein concentration in the eluate concentrated by centrifugation in protein concentrators (commercially available Amicon filters with a MWCO of 2 kDa) was under the detection limit of our protein quantification test and neither the crude extract nor the purified and concentrated protein showed any antimicrobial activity in our tests, as discussed in the
antimicrobial assays
section.

![results of SDS-Page from the first extraction](https://static.igem.org/mediawiki/2021/1/14/T--Tuebingen--results_coomassie_20210915.jpg)

*Figure 1: Results of SDS-PAGE with samples from the first extraction and purification.*
Samples from the plants infiltrated with the respective 3in1 vectors (c\_blank, c\_CHEN\_1, c\_CHEN\_6) were applied to the gel either directly as crude extract or after purification via Ni-NTA resin. The flowthrough of the column, the wash step and the elution fractions as well as the pooled samples after dialysis O.N. were also analyzed. The gel was stained with Coomassie-blue to detect protein bands. M-marker, FT-flow through, CE-crude extract, W-wash, a.d.-after dialysis, E-elution, 3in1 constructs labelled according to our official nomenclature

## Troubleshooting

### Overview

The empty blots and missing antimicrobial activity left us struggling with the interpretation of these results, as we could not identify which steps in the process did not work. Therefore, we collected different ideas and tried to test them to make our system work.

| The potential problem | How to test it |
| --- | --- |
| (A) Problem with the blotting procedure | Use a positive control, dot blot |
| (B) Expression of the peptide in plants did not work | No possibility to directly test expression, as all detection methods require extraction |
| (C) Expression of the AEP in plants did not work | Test AEP expression by western blot with HA antibody |
| (D) Low expression levels | Scale up |
| (E) Crushing of the leaves was not efficient enough | Not likely to be the problem |
| (F) Extraction buffer is not suitable for this peptide | Test more buffers |
| (G) The intramolecular His-tag cannot be recognized by the antibodies | MALDI to see a specific peak |
| (H) Peptide bound to plastic | Use control peptides in all steps and compare antimicrobial activity before |

### (A) Problem with the Blotting Procedure

#### A.1 The Potential Problem

Using a His-tagged protein as a positive control confirmed, that the blotting and detection procedure in general worked. However, as cyclotides being very small and cyclic are very special peptides, it is still possible that they behave differently, showing a different running behavior during electrophoresis or different association with the blotting membrane. It might therefore be possible that they are not blotted on the PVFD membrane under the standard blotting conditions we applied.

#### A.2 How to Test it

A dot blot would be a possible alternative to the ‘classical’ blotting technique as it is a more direct technique for detecting, analyzing, and identifying proteins. It manages without prior separation via SDS-PAGE and is therefore relatively easy and fast to perform. The sample is directly applied onto the membrane and then treated with the respective antibodies
[4](#res_ext_piaz)
. This method could give us the chance to determine the expression of our His-tagged cyclotide quite early in the process without the need for previously purified protein.

### (B) Expression of the Peptide in Plants Did Not Work

#### B.1 The Potential Problem

We used a GFP as an expression control to confirm the successful transfection of plant cells and the efficient gene expression of genes located on our vector. As we saw GFP fluorescence in our plant leaves, we know, that the agroinfiltration was successful and the plants expressed a protein from this vector with the very gene structure (same promoter and same terminator). However, we couldn’t detect the peptide neither as a specific band on the SDS-gel or in the western blot, nor detect its activity in the antimicrobial assays. Therefore, we had no direct proof for successful expression. Furthermore, it should be considered that GFP is a very well characterized and easy to express protein. Even if we codon-optimized the expression of the precursor and the AEP for
*Nicotiana*
, these genes remain more difficult to express, e.g. because of the ER-targeting signal. As our detection methods were all bound to a successful extraction of the peptide, we had no possibility to determine with step didn’t work. However, we assumed the extraction to be more likely to cause problems, as we could prove the expression of our control GFP.

### (C) Expression of the AEP in Plants Did Not Work

#### C.1 The Potential Problem

Similar to point (B), we also couldn’t directly prove the expression of the AEP. If the AEP wouldn’t be expressed (which is still unlikely as our GFP control worked, but not impossible), the cyclotide cannot be produced. The precursor would still be expressed, but run at a different size on the SDS-gel and most likely be more unstable and could therefore be degraded in the cell up to some extent.

#### C.2 How to Test it

As we tagged the AEP with a HA tag, we should be able to detect it in a western blot using a common anti-HA-antibody. Again, this detection would be limited by successful extraction of the enzyme, but as the AEP is a more “usual” protein than the cyclotide, extraction shouldn’t be as problematic.

### (D) Low Expression Levels

#### D.1 The Potential Problem

Another possibility is, that our whole system worked in general, but the expression levels of our peptide were too low for detection and antimicrobial activity.

#### D.2 How to Test it

We addressed this problem by increasing the number of leaves used for extraction and the assays, but we can never exclude that even higher numbers would lead to positive results. However, if the system would require such a high amount of plant mass, it wouldn’t be suitable for our project anyways, as infiltration of too many plants would contradict our demand for a fast and easy to handle system.

Click
[here](#res_ext_adba)
to jump to our further experiments testing this potential problem.

### (E) Crushing of the Leaves Was Not Efficient Enough

#### E.1 The Potential Problem

The next step in our protocol was the crushing of the leaves with mortar and pestle in liquid nitrogen. As our cyclotide should be located in the vacuole, successful extraction was bound to the destruction of the plant cell wall in this step. However, this step is very unlikely to be the problem in this process, this is believed to be the most efficient method to destroy the cells for protein extraction when compared to ribolyser tissue homogenizer or juicer, as confirmed by different experts we talked to. Therefore, we excluded this step to be the problem.

### (F) Extraction Buffer is Not Suitable for This Peptide

#### F.1 The Potential Problem

Even if it was shown, that the buffer we used was suitable to extract plant proteins
[2](#res_ext_potu)
, we assumed the buffer to be the most likely problem in our case, as the extraction of small peptides can be problematic and we had no information, how our grafted sequences could influence the process.

#### F.2 How to Test it

To adress this problem, more buffer conditions can be tested. Supplementing the HEPES extraction buffer with Tween20, was shown to help to extract more protein from plant tissue
[5](#res_ext_giri)
. An acidic acetonitrile-based extraction buffer has been shown to be a well performing method for the extraction of cyclotides
[6](#res_ext_poon)
. Therefore, we used two additional buffers, namely Lämmli buffer and an acidic acetonitrile-based buffer. During our excursion to Nomad, we got the advice, to only continue with the purification after we detected our cyclotide with Lämmli buffer extraction. Lämmli buffer is the SDS-PAGE gel loading buffer and typically contains 2% SDS and 5% β-mercaptoethanol, as well as 10% glycerol. Therefore, extraction in Lämmli buffer is a good first test to find bands on the gel and western blot, but the extracts are not very suitable for purification afterwards.

Click
[here](#res_ext_adca)
to jump to our further experiments testing this potential problem.

### (G) The Intramolecular His-tag Can Not Be Recognized by the Antibodies

#### G.1 The Potential Problem

We tagged our cyclotides with a His-tag for detection and purification. As the cyclotides are cyclic, the tag could not be located in the N- or C-terminal of the peptide, which would be the usual procedure. Instead, we located the six histidine residues in different loops for different constructs, flanked by flexible linkers to allow the tag as much structural freedom as possible. However, we found no comparative data for an intramolecular His-tag. Therefore, we do not know if the tag will be recognized by the antibodies in western blotting. Furthermore, the purification would be impossible, if the tag does not bind to the Ni-NTA resin. The intramolecular tag is one of the big uncertainty factors in our project. A non-functional tag would explain the problems in the western blot and purification.

#### G.2 How to Test it

The band in the SDS-gels should be visible independent of the tag, but only if the expression level is high enough. A further method to detect our peptide independent of the tag would be a MALDI-TOF MS experiment.

Click
[here](#res_ext_adda)
to jump to our further experiments testing this potential problem.

### (H) Peptide Bound to Plastic

#### The Potential Problem

Since the AMPs we used are positively charged they show strong affinity in binding non-specifically to surfaces via electrostatic forces
[7](#res_ext_wieg)
. Therefore, it is important to choose the right material of consumables in order to reduce the adsorption. A commonly used material for laboratory products is polystyrene which is negatively charged and thus our peptides would bind to the surface of these products
[7](#res_ext_wieg)
.

#### How to Test it

Before performing our experiments, we checked all our consumables we intended to use for working with the AMPs. For example, we bought special 96-well plates made of polypropylene. We also checked the material of reaction tubes and pipet tips which all turned out to be made of polypropylene. However, for some products we could not determine the material such as the material of the purification columns. In addition, we do not know how strong the binding of AMPs to negatively charged surfaces is and thus to what extend we would lose our product when working e.g., with materials made of polystyrene. To conclude, it might be possible that we lost part of our AMP-construct in the extraction and purification process. This would also explain why we could not detect our peptide in western blot. Due to lack of time, we could not do further experiments to prove this theory. But we thought about using a solution of our control peptides in all steps starting
from extraction until the end of purification. Finally, this sample could be used for antimicrobial assays. The comparison of the previous results of their antimicrobial activity with the solutions that undergo all process steps might reveal if the concentration of the AMP is decreasing during processing because the antimicrobial activity correlates to the concentration
[8](#res_ext_bern)
. This is still not a proof of this theory but might give some hints on what and where the issue is.

## Further Experiments

### Scaling up

We thought that the main problem in our first purifications was the low protein concentration resulting in a too low product concentration in the concentrated eluates. Therefore, 5-6 g of leaves were used in the next extraction and the crushed leaves were incubated in the buffer for 1 h to increase extraction efficiency. The total protein concentration in the extracts was now around 1.5 mg/ml (1.44-1.71 mg/ml). To load more proteins on the SDS-PAGE, 100 µl of each sample were precipitated with acetone before loading. On the Coomassie-stained SDS-PAGE, no additional band was observed in the cyclotide-expressing plant extracts when compared to the flow through after His-purification. After purification, no protein band was visible on the gel. In the western blot, no protein band was detected in neither the crude extracts nor the purified samples.

### Exchange of Buffer Components

#### Addition of Tween20

As already mentioned, from all the possibilities we figured out that could not have worked during our extraction and purification process, we considered the composition of our extraction buffer to be the most likely one. Therefore, we started a systematic testing process of different conditions. First, we supplemented our HEPES extraction buffer with Tween20, as this should help to extract more protein from plant tissue
[5](#res_ext_giri)
. However, the addition of 0.05% Tween20 to our extraction buffer led to the same result on SDS-PAGE and western blot as before. Therefore, we used three additional buffers, namely Lämmli buffer, an acidic acetonitrile-based buffer and an adjusted SDS-buffer.

#### Lämmli Buffer

We resuspend our in liquid nitrogen crushed plant extracts in Lämmli buffer. After a short incubation on ice, the samples were denaturated by heating to 95 °C, followed by direct application on an SDS-PAGE. All samples showed clearly more distinct bands of all kDa sizes on the Coomassie-stained SDS-gel in comparison to the prior gels, as visible in figure 2. For example, the bands in all lanes at the size of about 53 kDa are most likely from the large subunit of the enzyme Rubisco (Ribulose bisphosphate carboxylase), a plant enzyme involved in photosynthesis.

![results of SDS-PAGE, different extraction methods](https://static.igem.org/mediawiki/2021/a/a8/T--Tuebingen--results_coomassie_2021_09_29.jpg)

*Figure 2: Results of SDS-PAGE with samples from different extraction methods.*
The leaves transfected with the respective constructs as indicated in the figure were either extracted using Lämmli buffer or with an acidic acetonitrile-based buffer. As negative controls we used untransfected plant material as well as only transfected with vector p19. The gel was stained with Coomassie-blue after running. M-marker, untr.-untransfected, p19-control vector, 2in1 and 3in1 constructs labelled according to our official nomenclature

![western blot of His-tagged proteins](https://static.igem.org/mediawiki/2021/0/03/T--Tuebingen--results_western_antiHis_2021_09_29_5min.jpg)

*Figure 3: Results of the western blot for specific detection of His-tagged proteins.*
The separated protein bands on the SDS-PAGE gel of the samples extracted via Lämmli or Acetonitrile-based buffer were blotted onto a PVDF membrane for further analysis. The membrane was blocked in 5% milk/TBS-T and afterwards O.N. incubated with an anti-His-Tag primary antibody. After incubation with the corresponding secondary antibody the signal was detected after five minutes of exposure time. M-marker, untr.-untransfected, p19-control vector, 2in1 and 3in1 constructs labelled according to our official nomenclature, His-GlgB-positive control (~ 84 kDa)

In addition, we performed a western blot of the very same samples with an anti-His-Tag primary antibody to be able to specifically detect our His-tagged cyclotides. An immunoblot signal at a size of about 18 kDa could be detected (figure 3), which raised the suspicion that cyclization of our cyclotides by the AEP did not work, as the precursor of our constructs is about the same size as the detected signal.

Another possible explanation is that the band shows indeed our cyclotide, which does not run at the expected molecular range in the SDS-PAGE. This could be due to its highly positive charge, which might not be completely masked by the negatively charged SDS or due to its cyclic structure. Unfortunately, no signal was detected for our positive control, a His-tagged protein of about 84 kDa. This could be explained by the huge size difference between this control and our peptide. Thus, it could have happened that the blotting of the control on the membrane did not work.

#### Acidic acetonitrile-based Buffer

The results from the leaves, which were extracted using an acidic-acetonitrile based buffer are also displayed on the gel and blot (Figure 2 and 3). For this method (adapted from Poon et al, 2018)
[6](#res_ext_poon)
we used dried leaf material and evaporated the extraction solvent in the last step. Prior to applying these samples onto the SDS-PAGE some of the extract was therefore solubilized in ddH2O and then mixed with 2X Lämmli buffer in equal ratio. As can be seen in figure 2, the lanes of these samples show an irregular picture, as no clear and distinct bands can be identified apart from one apparently quite big band at the height of 3.5 kDa. For these samples no signal could be detected in the western blot (figure 3).

Because of the unusual bands in the gel, we decided to repeat this experiment and applied freshly prepared samples onto a Tricine-SDS gel as well as performed a western blot afterwards. The gel looks very similar to the first attempt (compare figure 2 and 4) but on the blot we were able to detect some signal this time (figure 5). However, the molecular weight (MW) range (smaller than 3.5 kDa) in which the signal occurs does not correspond to anything we expect. Only in the lane of the construct 3in1 c\_CHEN\_6 a weak band can be observed at the MW of about 8 kDa, which would be around the expected size of our cyclotide construct. But as the signal is very low, this assumption would have to be verified by further experiments.

![results of SDS-PAGE, acetonitrile-buffer](https://static.igem.org/mediawiki/2021/a/a5/T--Tuebingen--results_coomassie_2021_10_06_10er_acn.jpg)

*Figure 4: Results of SDS-PAGE with samples extracted with acidic acetonitrile-based buffer.*
The leaves transfected with the respective constructs as indicated in the figure were extracted with an acidic acetonitrile-based buffer. As negative controls we used only transfected with vector p19. The gel was stained with Coomassie-blue after running. M-marker, p19-control vector, 2in1 and 3in1 constructs labeled according to our official nomenclature, His-PirC-control protein (~ 14 kDa).

![western blot of His-tagged proteins](https://static.igem.org/mediawiki/2021/0/01/T--Tuebingen--results_western_antiHis_2021_10_07_10er_acn.jpg)

*Figure 5: Results of the western blot for specific detection of His-tagged proteins.*
The separated protein bands on the SDS-PAGE gel of the samples extracted with acidic acetonitrile-based buffer were blotted onto a PVDF membrane for further analysis. The membrane was blocked in 5% milk/TBS-T and afterwards O.N. incubated with an anti-His-Tag antibody, which was directly coupled to horseradish peroxidase for detection. The signal was detected after 30 seconds of exposure time. M-marker, p19-control vector, 3in1 constructs labelled according to our official nomenclature, His-PirC-control protein (~ 14 kDa)

#### Adjusted SDS-buffer

From the above stated results, we concluded that the extraction with a buffer supplemented with SDS seems to be the best approach. However, samples dissolved in Lämmli buffer are unsuitable for further experiments, neither purification nor assays to be able to verify the antimicrobial activity. Thus, we decided to use a slightly adjusted (only 1% SDS and no β-mercaptoethanol as reducing agent) Tris-based SDS buffer, which is recommended for the extraction of plant proteins for further use in protein quantification and analysis
[9](#res_ext_conl)
.

To use the peptides extracted with this method for our antimicrobial activity assays, we needed to remove the SDS. Our first attempt was to use dialysis chambers (which we originally used to remove the imidazole after purification via Ni-NTA resin) and dialyze against a large volume of HEPES buffer O.N. as this was the buffer all previous samples and our control peptides were solubilized in (compare
antimicrobial assays
section for more information). For analysis on the SDS-PAGE we took samples prior to and after dialysis. We could show that no remarkable amount of protein was lost (figure 6). Afterwards we figured out that removal of SDS via dialysis is somehow impossible due to the interaction of the anionic detergent with cationic proteins and we followed another protocol for the removal via acetone precipitation
[10](#res_ext_hend)
. Afterwards the protein pellets were resolubilized and analyzed on a gel (figure 7). We observed less bright bands here, however, this might have other reasons than loss of protein, e.g., less good staining.

![results of SDS-PAGE, adjusted SDS-buffer/dialysis](https://static.igem.org/mediawiki/2021/d/db/T--Tuebingen--results_coomassie_2021_10_06_15er.jpg)

*Figure 6: Results of SDS-PAGE with samples from extraction with adjusted SDS-buffer before and after dialysis.*
The leaves transfected with the respective constructs as indicated in the figure were extracted using the adjusted SDS-buffer and either applied before or after dialysis O.N. As negative controls we used plant material only transfected with control vector p19. As a positive control for the following western blot, we used a His-tagged protein. The gel was stained with Coomassie-blue after running. M-marker, p19-control vector, 3in1 constructs labelled according to our official nomenclature, His-PirC-control protein (~ 14 kDa), extr.-extraction, a.d.-after dialysis

![results of SDS-PAGE, adjusted SDS-buffer/precipitation](https://static.igem.org/mediawiki/2021/8/80/T--Tuebingen--results_coomassie_2021_10_06_10er.jpg)

*Figure 7: Results of SDS-PAGE with samples from extraction with adjusted SDS-buffer after precipitation.*
The leaves transfected with the respective constructs as indicated in the figure were extracted using the adjusted SDS-buffer, dialysed O.N. against HEPES buffer and afterwards precipitated via acetone precipitation for removal of SDS from the samples. As negative controls we used plant material only transfected with control vector p19. The gel was stained with Coomassie-blue after running. M-marker, p19-control vector, 2in1 and 3in1 constructs labelled according to our official nomenclature, precip.-precipitated samples

![western blot of His-tagged proteins](https://static.igem.org/mediawiki/2021/7/73/T--Tuebingen--results_western_antiHis_2021_10_07_15er.jpg)

*Figure 8: Results of the western blot for specific detection of His-tagged proteins.*
The separated protein bands on the SDS-PAGE gel of the samples extracted with adjusted SDS-buffer were blotted onto a PVDF membrane for further analysis. The membrane was blocked in 5% milk/TBS-T and afterwards O.N. incubated with an anti-His-Tag antibody, which was directly coupled to horseradish peroxidase for detection. As a positive control, we used a His-tagged protein. The signal of His-PirC and the right marker was detected after five seconds of exposure time. The signal of the remaining blot was detected after five minutes of exposure time with the positive control covered. M-marker, p19-control vector, 3in1 constructs labelled according to our official nomenclature, His-PirC-control protein (~ 14 kDa), extr.-extraction, a.d.-after dialysis

Here we also used western blotting as a further method for analysis of the separated protein bands on the associated gels. First of all, it can be mentioned that this time we were able to see a signal of our positive control, a His-tagged protein with a size of about 14 kDa. The protein sample was unfortunately not really purified and therefore bands of other molecular weight can also be observed (figure 8). Furthermore, on the blot of the gel with the samples directly after extraction and after dialysis O.N. several bands are visible at a size of about 18 kDa and 24 kDa. As these bands are also detectable in the negative control (p19), it might be unspecific binding of the antibody. The western blot of the gel with the samples after SDS removal via precipitation showed no signal except for the positive control (data not shown).

### MALDI-TOF MS

As stated above, plant crude extracts with Lämmli buffer and with acidic acetonitrile-based buffer showed protein bands with questionable identity: The 18 kDa band detected with an anti-His antibody in the Lämmli extracts, which may correspond to the cyclotide precursor or could indicate an unusual SDS-PAGE running pattern of the cyclotide itself, and the diffuse 3.5 kDa protein load visible in the Coomassie-stained acetonitrile extracts. To get more insights into the identity of these bands, mass spectrometry (MS) was performed.

#### Procedure

At first, gel slices at the designated height, corresponding to the protein MW marker, were cut out of the Coomassie-stained SDS-PAGE gels. For the Lämmli extracts, gel slices were cut out at the 18 kDa height, while for the acetonitrile extracts, gel slices were cut out at the 3.5 kDa height.

Subsequently, the gel slices were destained, homogenized, extracted and dried from solvent in a vacuum concentrator. The dried samples were dissolved in 1:10 diluted HEPES extraction buffer, together with dried acetonitrile extracts. These samples were measured using MALDI-TOF MS.

#### Results

Most of the samples’ MS spectra showed no peaks with appropriate m/z ratio with relevant peak intensity. The MS spectrum of the 18 kDa Lämmli extract gel slice of 3in1 construct c\_CHEN\_1 showed a single peak corresponding to a m/z ratio of 5724.41 (figure 9). This value could fit with a single-charged M+ ion of cyclic c\_CHEN\_1, which has a theoretical average mass of 5718.30 Da with all cysteine (cys) residues reduced, as would be expected after the SDS-PAGE involving the reducing agent β-mercaptoethanol, and a theoretical average mass of 5712.30 Da with all cys residues oxidized. The deviation between theoretical mass and observed m/z ratio could be explained by the fact that the used mass spectrometer was only calibrated in the range of 2 to 4 kDa. Furthermore, it is unknown in which form our peptide occurs, since unknown modifications might be adhered. However, the running pattern in the SDS-PAGE gel does not fit the MW of our cyclotide
construct. We speculated that the high positive charge of our construct, originating from its His6 tag and the cationic AMP graft, could influence its SDS running pattern.

![MALDI-TOF MS spectrum](https://static.igem.org/mediawiki/2021/6/6f/T--Tuebingen--results_maldi_3in1_3_precursorfromgel_remaininggelpiecesupernatant.jpg)

*Figure 9: MALDI-TOF MS spectrum of an 18 kDa gel slice of 3in1 construct c\_CHEN\_1, detected in a m/z range of 1000 to 7000.*
The gel slice was previously destained, protein extracted and dried from extraction solvent.

The dissolved acetonitrile extracts were additionally concentrated and desalted using C18 resin. The MALDI-TOF MS spectrum of 2in1 construct c\_CHEN\_6 showed no fitting peaks. However, many peaks corresponding to m/z ratios around 3400 were visible (figure 10 A). These peaks fit single-charge M+ ions of peptides making up the broad band visible at around 3.5 kDa in the Coomassie-stained SDS-PAGE gel of the acetonitrile extracts. Therefore, we concluded that our cyclotides are unlikely to be represented by this 3.5 kDa band. The spectrum of 3in1 construct c\_CHEN\_6 showed a low-intensity peak with a m/z ratio of 6033.16 (figure 10 B). This value is similar to the average mass of a single-charged M+ ion of cyclic c\_CHEN\_6, which is expected to be 6041.89 Da with all cys residues reduced, and 6035.89 Da with all cys residues oxidized, as would be expected since no reducing agent was used on this construct.

![MALDI-TOF MS spectrum](https://static.igem.org/mediawiki/2021/3/38/T--Tuebingen--results_maldi_ACNextr.jpg)

*Figure 10: MALDI-TOF MS spectra of (A) acetonitrile-based extract of 2in1 construct c\_CHEN\_6, detected in a m/z range of 1000 to 7000, and (B) acetonitrile-based extract of 3in1 construct c\_CHEN\_6, detected in a m/z range of 4000 to 6500.*
Both samples were previously concentrated and desalted using C18 resin.

Additionally, LC-MS was performed with the concentrated and desalted acetonitrile extracts of 2in1 and 3in1 construct c\_CHEN\_6. However, we were not able to detect any peaks characteristic for our cyclotide constructs (data not shown).

#### Conclusion

All in all, MALDI-TOF MS was able to confirm the presence of peptides with masses highly similar to the cyclic constructs c\_CHEN\_1 and c\_CHEN\_6, occurring in the Lämmli extract or in the acetonitrile extract, respectively. However, it is difficult to draw the conclusion that we actually detected our cyclotides, since the MS was performed with crude extracts most likely containing thousands of different peptides and proteins. We therefore presume that we may have actually detected our cyclotides using MALDI-TOF MS and western blot, but that peptide concentration is too low to see them in the LC-MS spectrum. In general, western blot is a method better suited for the detection of low abundance peptides, while mass spectrometry-based methods are used to characterize the exact mass of proteins already proven to be present
[11](#res_ext_asla)
. Also, the occurrence of only a single peak corresponding to the cyclotide constructs in the mass spectra fits previous MALDI-TOF results of cyclotides purified from
*N. bentamiana*
.

# References

1
Promega Corporation: HisLink Protein Purification Resin Technical Bulletin #TB327.

2
Potula, H. H. Surya Kumar; Kathuria, Sonal Roy; Ghosh, A. K.; Maiti, T. K.; Dey, S. (2008): Transient expression, purification and characterization of bioactive human fibroblast growth factor 8b in tobacco plants. In: Transgenic research 17 (1), S. 19–32. DOI: 10.1007/s11248-007-9072-4.

3
Schägger, H. (2006). Tricine-SDS-PAGE. Nature Protocols, 1(1), 16–22. https://doi.org/10.1038/nprot.2006.4

4
Piazza, R. M., Caetano, B. A., Henrique, C. P., Luz, D., Munhoz, D. D., Polatto, J. M., Rocha, L. B., Silva, M. A., & Mitsunari, T. (2020). Chapter 6 - Immunological tests for diarrhoea caused by diarrhoeagenic Escherichia coli targeting their main virulence factors. In C. S. Pavia & V. Gurtler (Eds.), Methods in Microbiology: Immunological Methods in Microbiology (Vol. 47, pp. 151–207). Academic Press. https://doi.org/10.1016/bs.mim.2019.11.004

5
Giritch, Anatoli; Marillonnet, Sylvestre; Engler, Carola; van Eldik, Gerben; Botterman, Johan; Klimyuk, Victor; Gleba, Yuri (2006): Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors. In: Proceedings of the National Academy of Sciences 103 (40), S. 14701–14706. DOI: 10.1073/pnas.0606631103.

6
Poon, S., Harris, K. S., Jackson, M. A., McCorkelle, O. C., Gilding, E. K., Durek, T., van der Weerden, N. L., Craik, D. J., & Anderson, M. A. (2018). Co-expression of a cyclizing asparaginyl endopeptidase enables efficient production of cyclic peptides in planta. Journal of Experimental Botany, 69(3), 633–641. https://doi.org/10.1093/jxb/erx422

7
Wiegand, Irith; Hilpert, Kai; Hancock, Robert E. W. (2008): Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. In: Nature protocols 3 (2), S. 163–175. DOI: 10.1038/nprot.2007.521.

8
Bernier, Steve P.; Surette, Michael G. (2013): Concentration-dependent activity of antibiotics in natural environments. In Frontiers in microbiology 4, p. 20. DOI: 10.3389/fmicb.2013.00020.

9
Conlon, H. E., & Salter, M. G. (2007). Plant Protein Extraction. In E. Rosato (Ed.), Circadian Rhythms: Methods and Protocols (pp. 379–383). Humana Press. https://doi.org/10.1007/978-1-59745-257-1\_28

10
Henderson, L. E., Oroszlan, S., & Konigsberg, W. (1979). A micromethod for complete removal of dodecyl sulfate from proteins by ion-pair extraction. Analytical Biochemistry, 93, 153–157. https://doi.org/10.1016/S0003-2697(79)80129-3

11
Aslam, B., Basit, M., Nisar, M. A., Khurshid, M., & Rasool, M. H. (2017). Proteomics: Technologies and Their Applications. Journal of Chromatographic Science, 55(2), 182–196. https://doi.org/10.1093/chromsci/bmw167

Go To

[Antimicrobial Assays](#res_ass_anti)
[Inhibition zone assays](#res_ass_inhi)
[Microdilution assays](#res_ass_micr)
[References](#res_ass_refe)

# Antimicrobial Assays

* *Microdilution assays could be shown to be a suitable method for verification of antimicrobial activity.*
* *No significantly antimicrobial activity of our cyclotides could be shown.*

## Inhibition zone assays

As an easy and fast method to estimate the antimicrobial activity of our antimicrobial peptides (AMPs) we used inhibition zone assays. This method, also known as Agar disk-diffusion testing, was developed in 1940
[1](#res_ass_heat)
and is nowadays still the official standard method in many clinical microbiology laboratories
[2](#res_ass_clsi)
. Generally, agar plates are inoculated with a known concentration of test microorganism and then filter paper discs containing the compound to be tested are placed on the agar. The germination and growth of bacteria is inhibited as the antimicrobial substance diffuses into the agar and as a read out the diameters of inhibition growth zones are measured
[3](#res_ass_balo)
.

Therefore,
*Escherichia coli (E. coli)*
BW25113 and
*Bacillus subtilis (B. subtilis)*
168 were grown as overnight cultures in LB medium and those were used to inoculate new cultures. When they reached middle logarithmic growth phase (OD
600
= 0.8 - 1.0) they were diluted either 1:10 or 1:50 and spread onto LB-agar plates. Small filter plates were placed onto the agar plates and 20 µl of sample were pipetted onto each plate. The plates were incubated over night at 37 °C.

We used the crude extract of the transfected
*Nicotiana benthamiana*
leaves as well as the purified peptides for testing. As positive controls we applied the chemically synthesized AMPs CHEN and KR-12 as these are grafted into our cyclotide construct. We also tested leaves from plants that were transfected with the construct of the empty cyclotide scaffold (c\_blank) as a negative control. All samples were solubilized in HEPES buffer (100 mM HEPES 150 mM NaCl, 3 mM KCl, pH 7.5), thus an influence of the buffer system can be ruled out.

After incubation of the agar plates O.N. we observed inhibition zones around the filter plates, on which on our positive controls were applied for both bacterial strains, which is shown in figure 1 and figure 2, respectively. However, we used much higher concentrations (100 µM and 10 mM) than the minimal inhibitory concentration (MIC) that can be found in the literature for the respective AMPs (MIC
CHEN
 = 3.13 µM, MIC
KR-12
 = 2.5 µM)
[4,](#res_ass_dong)
[5](#res_ass_guna)
. Neither the crude extracts of our infiltrated
*N. benthamiana*
leaves nor the purified peptides to be tested show any inhibition of bacterial growth. The negative control shows no inhibition as well.

The fact that even our positive controls only worked quite high concentrated indicates that the inhibition zone assay is unsuitable for the determination of the antimicrobial activity of AMPs. This might be due to interactions with the LB medium, which might shield the activity of the negatively charged AMPs as this charge is crucial for their functionality. This has been observed by other researchers before
[6](#res_ass_wieg)
. Therefore, we decided not to use these assays anymore for the testing of our peptides but focused on microdilution assays.

![inhibition zone assay](https://static.igem.org/mediawiki/2021/9/9a/T--Tuebingen--results_assays-fig1.jpg)

*Figure 1: Inhibition zone assay with 1:10 diluted B. subtilis.*
Of a
*B. subtilis*
culture in middle-logarithmic growth phase diluted 1:10 200 µl were spread on LB-agar plates. Of each sample 20 µl were pipetted onto the filter plates. Samples were applied as indicated in the description. CE - crude extract, purified - purified via IMAC, 1:50 - samples were diluted 1:50 prior application

![inhibition zone assay](https://static.igem.org/mediawiki/2021/a/ae/T--Tuebingen--results_assays-fig2.jpg)

*Figure 2: Inhibition zone assay with 1:10 diluted E. coli.*
Of an
*E. coli*
culture in middle-logarithmic growth phase diluted 1:10 200 µl were spread on LB-agar plates. Of each sample 20 µl were pipetted onto the filter plates. Samples were applied as indicated in the description. CE - crude extract, purified - purified via IMAC, 1:50 - samples were diluted 1:50 prior application

## Microdilution assays

Two-fold microdilution assays were performed in order to obtain quantitative results. In each well of a 96-well plate, bacterial suspension of
*E. coli*
or
*B. subtilis*
overnight cultures were aliquoted. Samples were added in a two-fold diluting manner and plates were incubated at 37°C for 24h. Before (t
0
)and after (t
1
) incubation, OD
600
was measured. We calculated bacterial growth with the following equation: t
1
- t
0
. Since all data are represented as delta t, the influence of intrinsic colors of extracts can be excluded. For cultivation we used brain heart infusion (BHI) and the standard medium for microdilution assays, Mueller-Hinton Broth (MH). However, antimicrobial activity might be hidden by media components
[6](#res_ass_wieg)
. Therefore, we diluted MH 1 to 5 (MH 1/5) in order to reduce the influence of media on our constructs, as already shown in a recent publication
[7](#res_ass_koeh)
.

Since we used our crude extracts, we could not determine the concentration of the constructs. Thus, all results are stated as dilution factors of the respective crude extracts. For each sample we performed a serial dilution series starting with the undiluted extract (=1) until a 1/32 (=0,03) dilution. As growth controls we cultivated bacteria in the respective media without any extracts. We used synthesized peptides KR-12 und CHEN diluted in HEPES buffer (100 mM HEPES 150 mM NaCl, 3 mM KCl, pH 7.5) in defined concentrations for positive controls. All conditions were prepared in triplicates and results are stated as mean of each triplicate. Statistical analysis was performed in Origin 2019 using 1-way ANOVA with p-value at 0.05.

![results of a microdilution assay, MH](https://static.igem.org/mediawiki/2021/8/84/T--Tuebingen--results_assays-fig3.jpg)

*Figure 3: Two-fold microdilution assay in MH medium with E. coli and B. subtilis incubating in control peptides KR-12 and CHEN.*
*E. coli*
shows no statistical reduction (p=0.05) in growth for both AMPs compared to the growth control (concentration = 0). Cell growth of
*B. subtilis*
decreased with increasing concentrations of CHEN but no statistical difference in cell growth was detected with KR-12.

As already assumed, different media show different effects of our constructs. This is due to the fact that some media conditions can hide the antimicrobial activity
[6,](#res_ass_wieg)
[7](#res_ass_koeh)
. Since we did not observe any activity in BHI, neither for our constructs nor for the control peptides, we excluded these results in the following discussion. In the control peptides, we did not observe growth inhibition of
*E. coli*
in MH medium. In contrast,
*B. subtilis*
is sensitive to the control peptides and bacterial cell growth is significantly reduced already with a 31,3 µM concentration (fig. 3). With the control peptides we observed in MH media diluted to 1/5 (MH 1/5) an inhibition in bacterial growth in both, E. coli and B. subtilis (fig. 4) However, the growth control of both bacteria strains in MH 1/5 showed lower cell growth compared to undiluted MH media (fig. 3-4). Therefore, it might be possible that bacteria are already stressed because of a lack in nutrition and this might make the cell more susceptible to the peptides. Interestingly, in all control samples, except for E. coli in MH 1/5, CHEN showed stronger antimicrobial activity compared to KR-12 (fig. 3-4).

![results of a microdilution assay, MH15](https://static.igem.org/mediawiki/2021/5/52/T--Tuebingen--results_assays-fig4.jpg)

*Figure 4: Two-fold microdilution assay in MH 1/5 medium with E. coli and B. subtilis incubating in control peptides KR-12 and CHEN.*
MH medium was diluted with sterile water to obtain a 1/5 dilution. Both bacteria strains show a minimal inhibitory concentration (MIC) of 125µM CHEN. MIC of KR-12 with E. coli is also 125µM while B. subtilis shows no total growth inhibition at the maximum concentration of 500µM KR-12.

For all undiluted crude extracts significant cell growth decrease was observed in all constructs for
*B. subtilis*
in MH media and in c\_CHEN\_1 construct for E. coli in MH compared to growth control (fig. 5). However, both bacteria strains showed an increase in cellular growth compared to growth control in MH 1/5. As a result, undiluted extracts have beneficial effects on bacteria growth in diluted MH media. These results are in contrast to the ones of the control peptides, where samples in MH 1/5 showed stronger antimicrobial activity. In full MH medium,
*B. subtilis*
showed in all constructs a significant growth reduction compared to the growth control. However, this is also the case for constructs without included AMPs. Therefore, other factors have an influence on cell growth. It is possible, that buffers or residuals of any process step inhibit bacterial growth.

While figure 5 represents all undiluted extracts of all different constructs, in figure 6 and 7 all tested dilutions of the constructs c\_blank, c\_CHEN\_1 and untransfected leaves can be found. Since CHEN showed stronger activity in the control peptides, we wanted to present this construct as a representative dilution series. Similar to the overview (fig. 5), constructs with CHEN showed no effect or an increase in cell growth. Interestingly, the untransfected leaves show significant reduction in cell growth of
*B. subtilis*
in MH 1/5. Since all extracts were processed in a similar way, it is unclear why extracts of untransfected leaves have antimicrobial activity while extracts with constructs do not show growth inhibition.

![overview of our microdilution results](https://static.igem.org/mediawiki/2021/3/38/T--Tuebingen--results_microdilutionassay_overview_no_dilution.jpg)

*Figure 5: Overview of two-fold microdilution assay in MH 1/5 medium with E. coli and B. subtilis incubating in undiluted crude extracts.*
MH medium was diluted with sterile water to obtain a 1/5 dilution. Crude extracts of plants transfected with the constructs p19, c\_CHEN\_1, c\_KR12\_1, c\_blank and c\_CHEN\_6 were tested using a two-fold microdilution assay. Statistical growth reduction was observed for B. subtilis in MH media.

To sum up, we found that the AMPs CHEN and KR-12 induce a reduction in cell growth in both,
*E. coli*
and
*B. subtilis*
in diluted MH medium. However, we could not find conclusive results for our constructs. Further analysis needs to be done, in order to determine the antimicrobial activity of the different extracts.

![results of a microdilution assay, MH, CE](https://static.igem.org/mediawiki/2021/d/db/T--Tuebingen--results_assays-fig6.jpg)

*Figure 6: Two-fold microdilution assay in MH medium with E. coli and B. subtilis incubating in different crude extracts.*
Crude extract of plants transfected with the constructs c\_blank, c\_CHEN\_1 and untransfected leaves were tested using a two-fold microdilution assay.

![results of a microdilution assay, MH15, CE](https://static.igem.org/mediawiki/2021/d/dd/T--Tuebingen--results_assays-fig7.jpg)

*Figure 7: Two-fold microdilution assay in MH 1/5 medium with E. coli and B. subtilis incubating in different crude extracts.*
MH medium was diluted with sterile water to obtain a 1/5 dilution. Crude extract of plants transfected with the constructs c\_blank, c\_CHEN\_1 and untransfected leaves were tested using a two-fold microdilution assay. In this specific experiment, data represents t
1
without subtracting t
0
.

# References

1
Heatley N. G. (1944). A method for the assay of penicillin. The Biochemical journal, 38(1), 61–65. https://doi.org/10.1042/bj0380061

2
CLSI, Performance Standards for Antimicrobial Disk Susceptibility Tests, Approved Standard, 7th ed., CLSI document M02-A11. Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA, 2012.

3
Balouiri, M., Sadiki, M., & Ibnsouda, S. K. (2016). Methods for in vitro evaluating antimicrobial activity: A review. Journal of Pharmaceutical Analysis, 6(2), 71–79. https://doi.org/10.1016/j.jpha.2015.11.005

4
Dong, W., Dong, Z., Mao, X., Sun, Y., Li, F., & Shang, D. (2016). Structure-activity analysis and biological studies of chensinin-1b analogues. Acta Biomaterialia, 37, 59–68. https://doi.org/10.1016/j.actbio.2016.04.003

5
Gunasekera, S., Muhammad, T., Strömstedt, A. A., Rosengren, K. J., & Göransson, U. (2020). Backbone Cyclization and Dimerization of LL-37-Derived Peptides Enhance Antimicrobial Activity and Proteolytic Stability. Frontiers in Microbiology, 11, 168. https://doi.org/10.3389/fmicb.2020.00168

6
Wiegand, Irith; Hilpert, Kai; Hancock, Robert E. W. (2008): Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. In Nature protocols 3 (2), pp. 163–175. DOI: 10.1038/nprot.2007.521.

7
Koehbach, Johannes; Gani, Jurnorain; Hilpert, Kai; Craik, David J. (2021): Comparison of a Short Linear Antimicrobial Peptide with Its Disulfide-Cyclized and Cyclotide-Grafted Variants against Clinically Relevant Pathogens. In Microorganisms 9 (6), p. 1249. DOI: 10.3390/microorganisms9061249.

# Model Results

## Accomplishments

1. We found hundreds of new potential cyclotides via genome mining.
2. We predicted the structures of our constructs.
3. During our partnership with iGEM team IISER Kolkata, we simulated the membrane interaction of our constructs and several AMPs.
4. We screened our constructs and several AMPs for their ability to bind to phospholipids.

[](https://static.igem.org/mediawiki/2021/0/08/T--Tuebingen--model-chen_cool_short.mp4)

Showcase: CHEN interacting with the membrane

## Further Information

To view the results for our Hidden Markov Model Search, Structure Prediction, Molecular Dynamics simulations, and Molecular Docking, please visit
[Model](https://2021.igem.org/Team%3ATuebingen/Model)
.

To get an idea on how we ran our simulations, please refer to
[Experiments](https://2021.igem.org/Team%3ATuebingen/Experiments)
.

### About Us

We are the iGEM Team Tuebingen, a group of motivated students who are working on creating a fast screening platform for stabilized peptides. We are aiming to provide a system that gives everyone the ability to stabilize peptides such as antimicrobial peptides to create better medical agents.

### Follow Us

### Contact Us

#### team@igem-tuebingen.de

#### +49 7071 9189 745

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![Affinity Designer](https://static.igem.org/mediawiki/2021/b/b7/T--Tuebingen--af_designer_icn_txt_ls_dark.png)

![Affinity Photo](https://static.igem.org/mediawiki/2021/3/39/T--Tuebingen--af_photo_icn_txt_ls_dark.png)

![Affinity Publisher](https://static.igem.org/mediawiki/2021/4/46/T--Tuebingen--af_publisher_icn_txt_ls_dark.png)

![Brand](https://static.igem.org/mediawiki/2021/3/3c/T--Tuebingen--brand.png)

![Greiner Bio One](https://static.igem.org/mediawiki/2021/d/d8/T--Tuebingen--greiner_bio_one.jpg)

![Promega](https://static.igem.org/mediawiki/2021/a/a5/T--Tuebingen--promega.png)

![SnapGene](https://static.igem.org/mediawiki/2021/2/2a/T--Tuebingen--snapgene.png)

![StuRa](https://static.igem.org/mediawiki/2021/7/7c/T--Tuebingen--stura.png)

![Toggl Plan](https://static.igem.org/mediawiki/2021/7/78/T--Tuebingen--toggl_plan.png)

![Twist Biosciences](https://static.igem.org/mediawiki/2021/c/cb/T--Tuebingen--twist_biosciences.png)

![Microsynth SEQLAB](https://static.igem.org/mediawiki/2021/0/0f/T--Tuebingen--microsynth.png)

![Corning](https://static.igem.org/mediawiki/2021/6/6e/T--Tuebingen--corning.png)

![Benchling](https://static.igem.org/mediawiki/2021/9/9e/T--Tuebingen--benchling.png)

![CMFI](https://static.igem.org/mediawiki/2021/6/6d/T--Tuebingen--cmfi.jpg)

![NewEngland Biolabs](https://static.igem.org/mediawiki/2021/3/34/T--Tuebingen--neb.png)

![IDT](https://static.igem.org/mediawiki/2021/b/b0/T--Tuebingen--idt.png)