iGEM 2020 — Team Heidelberg

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Heidelberg/Notebook

NAVIGATION

* [People](https://2020.igem.org/Team%3AHeidelberg/Team)
  + [Team](https://2020.igem.org/Team%3AHeidelberg/Team)
  + [Attributions](https://2020.igem.org/Team%3AHeidelberg/Attributions)
  + [Sponsors](https://2020.igem.org/Team%3AHeidelberg/Sponsoring)
* [Project](https://2020.igem.org/Team%3AHeidelberg/Description)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Description)
  + [RNA-Protein Interaction](https://2020.igem.org/Team%3AHeidelberg/RNA_Protein)
  + [PPR & Pumilio Domains](https://2020.igem.org/Team%3AHeidelberg/Pumilio_PPR)
  + [DNA:RNA Triple Helices](https://2020.igem.org/Team%3AHeidelberg/Triple_Helix)
  + [Split Trans Splicing Ribozymes](https://2020.igem.org/Team%3AHeidelberg/Trans_Splicing)
  + [Proposed Implementation](https://2020.igem.org/Team%3AHeidelberg/Implementation)
  + [Engineering Success](https://2020.igem.org/Team%3AHeidelberg/Engineering)
  + [Proof of Concept](https://2020.igem.org/Team%3AHeidelberg/Proof_Of_Concept)
  + [Contribution](https://2020.igem.org/Team%3AHeidelberg/Contribution)
  + [Achievements](https://2020.igem.org/Team%3AHeidelberg/Achievements)
* [Parts](https://2020.igem.org/Team%3AHeidelberg/Parts)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Parts)
  + [RBP Collection](https://2020.igem.org/Team%3AHeidelberg/Part_Collection)
* [Modeling](https://2020.igem.org/Team%3AHeidelberg/Model)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Model)
  + [RNA Operads](https://2020.igem.org/Team%3AHeidelberg/Model/RNA_Combinatorics)
  + [Modular RBP Molecular Modelling](https://2020.igem.org/Team%3AHeidelberg/Model/Pumilio_PPR)
* [Software](https://2020.igem.org/Team%3AHeidelberg/Software)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Software)
  + [PRISM](https://2020.igem.org/Team%3AHeidelberg/Software/PRISM)
  + [CoNCoRDE](https://2020.igem.org/Team%3AHeidelberg/Software/Concorde)
  + [3DOC](https://2020.igem.org/Team%3AHeidelberg/Software/3DOC)
  + [RISE](https://2020.igem.org/Team%3AHeidelberg/Software/RISE)
* [Human Practices](https://2020.igem.org/Team%3AHeidelberg/Human_Practices)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Human_Practices)
  + [Integrated Human Practices](https://2020.igem.org/Team%3AHeidelberg/Integrated)
  + [Expert Interviews](https://2020.igem.org/Team%3AHeidelberg/Interviews)
  + [Safety](https://2020.igem.org/Team%3AHeidelberg/Safety)
  + [Education](https://2020.igem.org/Team%3AHeidelberg/Education)
  + [Collaborations](https://2020.igem.org/Team%3AHeidelberg/Collaborations)
* [Notebook](https://2020.igem.org/Team%3AHeidelberg/Notebook)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Notebook)
  + [Methods](https://2020.igem.org/Team%3AHeidelberg/Experiments)
  + [Materials](https://2020.igem.org/Team%3AHeidelberg/Materials)
* [For Judges](https://2020.igem.org/Team%3AHeidelberg/Sitemap)

[The Legend of Cellda](https://2020.igem.org/Team%3AHeidelberg)

---

COLLAPSE NAVIGATION

---

* People
  + [Team](https://2020.igem.org/Team%3AHeidelberg/Team)
  + [Attributions](https://2020.igem.org/Team%3AHeidelberg/Attributions)
  + [Sponsors](https://2020.igem.org/Team%3AHeidelberg/Sponsoring)
* Project
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Description)
  + [RNA-Protein Interaction](https://2020.igem.org/Team%3AHeidelberg/RNA_Protein)
  + [PPR & Pumilio Domains](https://2020.igem.org/Team%3AHeidelberg/Pumilio_PPR)
  + [DNA:RNA Triple Helices](https://2020.igem.org/Team%3AHeidelberg/Triple_Helix)
  + [Split Trans Splicing Ribozymes](https://2020.igem.org/Team%3AHeidelberg/Trans_Splicing)
  + [Proposed Implementation](https://2020.igem.org/Team%3AHeidelberg/Implementation)
  + [Engineering Success](https://2020.igem.org/Team%3AHeidelberg/Engineering)
  + [Proof of Concept](https://2020.igem.org/Team%3AHeidelberg/Proof_Of_Concept)
  + [Contribution](https://2020.igem.org/Team%3AHeidelberg/Contribution)
  + [Achievements](https://2020.igem.org/Team%3AHeidelberg/Achievements)
* Parts
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Parts)
  + [RBP Collection](https://2020.igem.org/Team%3AHeidelberg/Part_Collection)
* Modeling
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Model)
  + [RNA Operads](https://2020.igem.org/Team%3AHeidelberg/Model/RNA_Combinatorics)
  + [Modular RBP Molecular Modelling](https://2020.igem.org/Team%3AHeidelberg/Model/Pumilio_PPR)
* Software
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Software)
  + [PRISM](https://2020.igem.org/Team%3AHeidelberg/Software/PRISM)
  + [CoNCoRDE](https://2020.igem.org/Team%3AHeidelberg/Software/Concorde)
  + [3DOC](https://2020.igem.org/Team%3AHeidelberg/Software/3DOC)
  + [RISE](https://2020.igem.org/Team%3AHeidelberg/Software/RISE)
* Human Practices
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Human_Practices)
  + [Integrated Human Practices](https://2020.igem.org/Team%3AHeidelberg/Integrated)
  + [Expert Interviews](https://2020.igem.org/Team%3AHeidelberg/Interviews)
  + [Safety](https://2020.igem.org/Team%3AHeidelberg/Safety)
  + [Education](https://2020.igem.org/Team%3AHeidelberg/Education)
  + [Collaborations](https://2020.igem.org/Team%3AHeidelberg/Collaborations)
  >
* Notebook
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Notebook)
  + [Methods](https://2020.igem.org/Team%3AHeidelberg/Experiments)
  + [Materials](https://2020.igem.org/Team%3AHeidelberg/Materials)
* [For Judges](https://2020.igem.org/Team%3AHeidelberg/Sitemap)

Notebook

Gel images and more

# Labbooks

To see what we did in the lab, take a look at the labbooks below.

[![Labbook_PPR_Proteins](https://static.igem.org/mediawiki/2020/6/68/T--Heidelberg--educationOverview.jpg)](https://static.igem.org/mediawiki/2020/2/29/T--Heidelberg--Labbook_PPR_Proteins.pdf)

[![Labbook_Split_GFP](https://static.igem.org/mediawiki/2020/6/68/T--Heidelberg--educationOverview.jpg)
Labbook\_Split\_GFP](https://static.igem.org/mediawiki/2020/8/8d/T--Heidelberg--Labbook_Split_GFP.pdf)

[![Labbook_Triple_Helix](https://static.igem.org/mediawiki/2020/6/68/T--Heidelberg--educationOverview.jpg)
Labbook\_Triple\_Helix](https://static.igem.org/mediawiki/2020/6/6a/T--Heidelberg--Labbook_Triple_Helix.pdf)

[![Labbook_Split_Trans_Splicing_Ribozymes](https://static.igem.org/mediawiki/2020/6/68/T--Heidelberg--educationOverview.jpg)
Labbook\_Split\_Trans\_Splicing\_Ribozymes](https://static.igem.org/mediawiki/2020/0/07/T--Heidelberg--Labbook_Split_Trans_Splicing_Ribozymes.pdf)

# Drylab

#### [Calendar Week 28](#openbox-1-collapse)

**3DOC**

A Python script based on PyRosetta was developed, which automatically combines PDBs of of RNA-binding Pumby Modules
for a user-inputted RNA sequence
Adamala2016ProgrammableRP
Chaudhury2010PyRosettaAS
. After repacking of mutated amino acids deviating from the Pumby consensus sequence as well as relax of the modules
connections, some problems involving wrong binding between the amino acid backbone remain.

**PRISM**

We discussed different neural network architectures and finally came to the idea to implement a GAN (generative
adversial network) for the generation of RNA binding proteins.

**RISE**
Under the impression of our discussions with other iGEM Teams at the [iGEM Meetup organized by the iGEM Team
Marburg](https://igemmarburg.com/meetup) we discussed in our team, how the iGEM Registry could become an even more valuable resource for our
team. For us it was clear that the database suffers from a lack of clarity and flexibility. Therefore, we asked
ourselves how we could improve the database.

**Collaboration with iGEM Manchester**
At the [iGEM Meetup
organized by the iGEM Team Marburg](https://igemmarburg.com/meetup) we got to know Miguel of the [iGEM Team Manchester](https://2020.igem.org/Team%3AManchester). We got into conversation about the iGEM
Registry, which we worked on later in the project RISE and agreed to meet some time later to exchange experiences.

#### [Calendar Week 29](#openbox-2-collapse)

**3DOC**

The Python script was refined by solving the issues based on wrong bindings between the amino acid backbones at the border of two Pumby modules. Work was started to expand the script on PPR Modules Coquille2014AnAP Barkan2012ACA.

**PRISM**

We started the development of PRISM discussing our ideas with [Prof. Gumbel](https://2020.igem.org/Team%3AHeidelberg/Integrated#Gumbel) from the Hochschule Mannheim. With him we discussed the principles of machine learning and which problems could arise, when applying large datasets to it. We identified the Swissprot database for pre-training of the Language Model Several databases, such as EcRBPome as well as RBPDP were reviewed for usage as data source for RNA-binding proteins in the finetuning step of the Language Model Consortium2019UniProtAW. One of the databases reviewed only offered the sequences rather than an identifier, which could be mapped to Uniprot. The entries of the other database could be mapped, but did not offer any RNA-motifs.

**CoNCoRDe**

We implemented Vienna RNA Inverse Fold as the first part of our approach developing CoNCoRDe. We hoped to solve RNA substructures easily and tested the method with the EteRNA100 benchmark dataset. Based on the findings that the algorithm included in VIenna RNA Inverse Fold does have a low computational cost, but has difficulties with harder RNA structures, we developed the idea of developing a deep learning network for RNA reinforcement learning.

#### [Calendar Week 30](#openbox-3-collapse)

**RISE**

Our plans to improve the iGEM Registry slowly became more concrete. We identified two keypoints as functionality for a better iGEM Database: Searching for entries not only based on the part name, but also the description and documentation. Filtering the iGEM Registry entries for uses and availability.

**CoNCoRDe**

Besides ViennaRNA we started implementing a deep learning network as a policy for RNA design based on reinforcement learning.

#### [Calendar Week 31](#openbox-4-collapse)

**PRISM**

In this week we also talked to [Prof. Giunchiglia](https://2020.igem.org/Team%3AHeidelberg/Integrated#Giunchiglia). Based on his feedback we revised eventual biases in the data we are using and refocussed our plans for PRISM on a classic Language model which is based on a modern transformer architecture Vaswani2017AttentionIA.

**RISE**

Our very first idea to build our team-internal tool, was to download the SQL Database, which can be downloaded via the iGEM Website. This plan failed. Not only didn’t we have a lot of experience with SQL Programming but also couldn’t we solve several issues concerning the Character encoding. We realized that accessing SQL database may not be a suitable way for teams with not a lot of programmatic experience.

**Collaboration with iGEM Manchester**

Following up our friendship we made in Calendar Week 29 with the iGEM Team Manchester we met the iGEM Team Manchester again and exchanged our experiences with iGEM Software development so far. iGEM Manchester asked for our advice, concerning their Wiki study project. We suggested implementing a web scraper and show off an example suitable for the iGEM Wiki page some time later.

**CoNCoRDe**

As reinforcement learning is relatively unstable and highly sensitive to hyperparameters, we added an advantage-weighted actor critic (AWAC) training algorithm, which already was implemented in the “torchsupport" library of our advisor Michael Jendrusch. We imported it from this library.

#### [Calendar Week 32](#openbox-5-collapse)

**3DOC**

The initial idea was developed further by expanding it to concatenate all types of proteins and protein domains directly to each other or via RNA linkers. For this a workflow to prepare DNA sequences and RNA linkers was developed to be used in Geneious Prime or for manual application.

**PRISM**

Also, several types of annotations relevant for the sequences were chosen: Gene Ontology (GO-Terms), Taxonomy, Total Charge, Amino Acid Prevalence, Amino Acid Types Prevalence and Ligand-Binding Consortium2019TheGOAshburner2000GeneOT. An automatic download of all databases used in the Language Model was implemented. This included the Swissprot data from Uniprot, Gene Ontology annotations as well as the Binding Database Gilson2016BindingDBI2.

**CoNCoRDe**

When we finalized our training in CoNCoRDe we got back into touch with our advisor Michael Jendrusch, as he has a lot of experience with RNA design and discussed with him our results of hyperparameter analysis and optimized the code.

#### [Calendar Week 33](#openbox-6-collapse)

**3DOC**

An automatic BLASTp-search on RCSB database for all protein sequences inputted into 3DOC and based on the BLAST-result automatic mutation and repacking of amino acid residues via PyRosetta were implemented to generate PDBs of the proteins and protein domains which should be fused or interconnected via a linker Berman2000ThePD. Some protein sequences showed after BLASTp-search a relatively low Bitscore and high E-value, so it is necessary to evaluate the accuracy of the proposed method for all types of proteins.

**PRISM**

The functions to generate vectors with all annotations for a specific sequence originating from Swissprot were established. For this all information from all databases were combined and interconnected. Also functionality to calculate further characteristics, such as the distribution of amino acid types in the sequence, was added. Last but not least padding for the sequences was introduced.

Both a high number of taxonomies and GO-Terms appear in the entries in Swissprot. Some of the GO-Terms and taxonomies only appear for a small number of proteins. To train the network efficiently several ways for feature reduction were overthought.

**RISE**

After some research on the iGEM Registry we decided to focus our efforts on the [point-in-time database dump](http://parts.igem.org/Registry_AP) provided on the iGEM website. In this week we implemented a first version of a .xml parser, which we want to extract all part information to build a .csv table or an own SQL database.

**Collaboration with iGEM Manchester**

We constantly stayed in contact with the iGEM Team Manchester concerning their Wiki Study project and their request to show them how to implement a web scraper to access an iGEM wiki. Following our first code examples we decided together with iGEM Team Manchester to support them in a more comprehensive way in their Wiki study project. We contacted [Benedict Wolf](https://2020.igem.org/Team%3AHeidelberg/Integrated#Wolf), who discussed with us the structure of iGEM Wiki pages and gave us advice how to optimize our web scraper.

**CoNCoRDe**

Based on the solved structures we implemented some optimizations of the CoNCoRDe algorithm.

#### [Calendar Week 34](#openbox-7-collapse)

**3DOC**

It was decided to implement trRosetta as an alternative to the proposed PyRosetta algorithm Yang2020ImprovedPS, when the E-Value exceeds 0.5 and the determined Bitscore is smaller than 100. For this hhblits, a method in the HH-suite3, got implemented as well to generate multiple sequence Alignments as input for trRosetta Steinegger2019HHsuite3FF.

**PRISM**

The Encoder and EncoderLayer as well as the Multi-Head Attention Mechanism were built based on the CTRL Transformer Model Architecture Keskar2019CTRLAC Vaswani2017AttentionIA. In this week we discussed the structure of the CTRL model with [Carola Fischer](https://2020.igem.org/Team%3AHeidelberg/Integrated#Fischer), who is a PhD student at the Technical University Berlin. Based on her feedback we started thinking how to validate our model.

It was decided to use Adam as an optimizer for our trainingKingma2015AdamAM. We used Adam with the betas = (0.9, 0.98). We also implemented a learning rate scheduler as proposed by Madani et al. In addition to that we decided to use 4000 steps as warmup steps for the training.

#### [Calendar Week 35](#openbox-8-collapse)

**RISE**

Finishing up our work on the XML parser we decided to insert all part information into a .csv file. By this, we can directly provide a low-entry-level file, which can already be accessed and manipulated by any table calculation program. We also decided to develop a Python program to access the csv upon user request.

**Collaboration with iGEM Manchester**

In our meeting with iGEM Manchester we presented to iGEM Manchester our work concerning analyzing the texts on iGEM Wikis with our web scraper. Refining the concept of the Wiki study iGEM Manchester suggested implementing more functions into the web scraper, such as a PDF and video analysis, which we worked on in the following weeks.

**PRISM**

The training function as well as the function for cross validation of the pretraining step of the neural network were implemented. In this week we started the pretraining of our model. Therefore we consulted Michael Jendrusch, our advisor, who gave us feedback on the model architecture. With the help of the [bwforcluster](https://www.urz.uni-heidelberg.de/de/bwforcluster), we could lower the high amount of computational cost. Parallel to some test runs first came to idea of hyperparameter search.

**CoNCoRDe**

Besides the heart of CoNCoRDe we decided to provide several accompanying tools to generate RNAs. These include the scripts “random\_structures.py" and “design\_simple.py".These scripts are supposed to generate RNA sequences with a fixed-length, but limited to user-inputted constraints and RNA sequences, which are conditioned on a given RNA secondary structure and sequence constraints. With the tool “design\_rna\_triple\_helix.py" we also included a tool to design the RNA for usage in a DNA-RNA triple helix. The development of these tools spread over the following weeks.

#### [Calendar Week 36](#openbox-9-collapse)

**3DOC**

The algorithm to interconnect the PDBs via the Amino Acid and RNA Linkers was implemented. Also, the input to 3DOC for RNA Linker PDBs was established. Setting up the RNA-denovo Protocol in PyRosetta was quite challenging. Therefore we propose to use the ROSIE FARFAR2 protocol instead Watkins2019FARFAR2ID
Lyskov2013ServerificationOM. Secondary structures were generated using ViennaRNA Lorenz2011ViennaRNAP2.

**RISE**

The implementation of the Python Program, which we baptized as our iGEM Registry intelligent Search Engine (RISE), took place in this and the following calender week. In the process of the development of the Python program, we also decided to implement it into a Jupyter Notebook together with [Julius Upmeier zu Belzen](https://github.com/quantopian/qgrid%3EQgrid%3C/a%3E%2C%20so%20the%20database%20can%20easily%20be%20filtered.%3Cbr%3E%3Cb%3EPRISM%3C/b%3E%3Cbr%3EBased%20on%20the%20feedback%20of%20Michael%20we%20decided%20to%20reduce%20the%20number%20of%20layers%20of%20the%20neural%20network%20and%20set%20the%20dimension%20of%20the%20network%20to%20512.%20For%20this%20we%20needed%20a%20feature%20reduction%20method%20as%20already%20mentioned%20in%20Week%2033.%20Finally%2C%20feature%20reduction%20was%20reached%20for%20the%20taxonomies%20and%20GO-Terms%20with%20filtering%20for%20the%20prevalence.%20The%20GO-Tools%20were%20further%20processed%20by%20adding%20RBP-specific%20GO-Terms%20and%20filtered%20with%20GOATOOLS%20through%20determining%20semantic%20similarities%20between%20GO-Terms%20and%20their%20function%20%3Cx-ref%3EKlopfenstein2018GOATOOLSAP%3C/x-ref%3E.%20For%20this%20we%20talked%20to%20%3Ca%20href%3D), who is an expert in GO-Terms and gave us feedback on how to reduce the number of GO-Terms by filtering and applying semantic similarity.

#### [Calendar Week 37](#openbox-10-collapse)

**PRISM**

We integrated the ATracT database into the preprocessing pipeline, so the datasets for fine-tuning were generated. Giudice2016ATtRACTaDO. For this we mapped the identifiers of the ATracT database to Uniprot. The RNA-motifs bound by the proteins were also one-hot encoded. We met again with [Carola Fischer](https://2020.igem.org/Team%3AHeidelberg/Integrated#Fischer), who is a PhD student at the Technical University Berlin, to further discuss the influence of hyperparameters on general and on the fine-tuning process. Based on her feedback we run different tests to optimize our fine-tuning.

At this week, we could finally start a training job. We analysed the loss and accuracy of our model over the steps. We recognized that our model is unstable. In order to improve the performance and the convergence speed of our model, we were searching for alternatives. We found the ReZero concept as an alternative approach of the transformer architecture and implemented it.

#### [Calendar Week 38](#openbox-11-collapse)

**3DOC**

We decided to provide two scripts for the RNA denovo and RNP-structure prediction with Rosetta. These scripts are supposed to prepare the file outputted by 3DOC for the application in Rosetta.

**PRISM**

The Rezero Transformer network was now implemented and we could start some new training processes. We observed a much more stable transformer.
In order to increase the performance of our model, we began to search for optimal hyperparameters and consulted Carola Fischer again.

#### [Calendar Week 39](#openbox-12-collapse)

**Collaboration with iGEM Manchester**

Until this week we implemented more functionality into the web scraper and discussed them with the iGEM team Manchester. iGEM Manchester showed off the draft of their analysis and results, which they built with the previous versions of our web scraper.

**PRISM**

We tried to vary our batch size but we early recognized some computational constraints. In order to avoid these problems we found a solution which is called gradient accumulation. We implemented it and it worked. We could now apply much bigger batch sizes as before. This was a huge milestone in our hyperparameter search.

#### [Calendar Week 40](#openbox-13-collapse)

**PRISM**

At this week we started several training jobs with different parameters as batch size and warmup steps. We also implemented a function which creates a confusion matrix for the validation of our model. We also created an user-friendly python script for generating sequences, so any judge and scientist interested in using the output of PRISM is enabled to do so.

**CoNCoRDe**

As our project deals with the \*in vivo\* expression of functional RNA, a common design task features the design of flanking Hammer-Head Ribozymes (HHR) for a given target RNA sequence. We have specifically provided a version of our `design\_simple.py` script specialized to this task. It designs flanking HHR for a sequence given just the 5' and 3' ends of that sequence.

#### [Calendar Week 41](#openbox-14-collapse)

**PRISM**

This week, we implemented several functions for the generation of new RBP-sequences. We implemented a function to generate a sequence based on a user-inputted RNA-motif and annotations vector and a function based on a random annotations vector and RNA-motif. First we had to discuss which method to use. We found some methods and discussed them based on the paper “The Curious Case of Neural Text Degeneration" Holtzman2020TheCC. In addition to that we consulted Michael Jendrusch again. He gave us the tip to use temperature controlled greedy sampling. The functions were written and tested on random data.

**Collaboration with iGEM Manchester**

In our final meeting with iGEM Manchester we presented our final web scraper to the iGEM team Manchester and discussed with them the first results of their analysis.

#### [Calendar Week 42](#openbox-15-collapse)

**PRISM**

We started the final trainings. The models were saved as pytorch state dicts and used for our fine-tuning. All weights and biases were freezed but the embedding layer of our protein sequences and prediction layer were unfreezed for finetuning. We generated in total ten test sequences with our model, checked them via a BLAST search and inputted them into 3DOC respectively trRosetta.

#### [Calendar Week 43](#openbox-16-collapse)

Of course, our experiments all have been completed until now, our work already was well documented on our wiki, and the wiki itself looked bright and shiny. If this would not have been a dream overall, we would have relaxed in Week 43.

### References

#### Quote

*“”*

#### Useful **Links**

[Special pages](https://igem.org/Special%3ASpecialPages)

[Main Page](https://igem.org/Main_Page)

#### Follow us on

[*Instagram*](https://www.instagram.com/igem.heidelberg/)
&
[*Twitter*](https://twitter.com/igemHD)

#### Contact us

igemhd
@protonmail.com

Im Neuenheimer Feld 267

69120 Heidelberg

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Heidelberg/Engineering

NAVIGATION

* [People](https://2020.igem.org/Team%3AHeidelberg/Team)
  + [Team](https://2020.igem.org/Team%3AHeidelberg/Team)
  + [Attributions](https://2020.igem.org/Team%3AHeidelberg/Attributions)
  + [Sponsors](https://2020.igem.org/Team%3AHeidelberg/Sponsoring)
* [Project](https://2020.igem.org/Team%3AHeidelberg/Description)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Description)
  + [RNA-Protein Interaction](https://2020.igem.org/Team%3AHeidelberg/RNA_Protein)
  + [PPR & Pumilio Domains](https://2020.igem.org/Team%3AHeidelberg/Pumilio_PPR)
  + [DNA:RNA Triple Helices](https://2020.igem.org/Team%3AHeidelberg/Triple_Helix)
  + [Split Trans Splicing Ribozymes](https://2020.igem.org/Team%3AHeidelberg/Trans_Splicing)
  + [Proposed Implementation](https://2020.igem.org/Team%3AHeidelberg/Implementation)
  + [Engineering Success](https://2020.igem.org/Team%3AHeidelberg/Engineering)
  + [Proof of Concept](https://2020.igem.org/Team%3AHeidelberg/Proof_Of_Concept)
  + [Contribution](https://2020.igem.org/Team%3AHeidelberg/Contribution)
  + [Achievements](https://2020.igem.org/Team%3AHeidelberg/Achievements)
* [Parts](https://2020.igem.org/Team%3AHeidelberg/Parts)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Parts)
  + [RBP Collection](https://2020.igem.org/Team%3AHeidelberg/Part_Collection)
* [Modeling](https://2020.igem.org/Team%3AHeidelberg/Model)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Model)
  + [RNA Operads](https://2020.igem.org/Team%3AHeidelberg/Model/RNA_Combinatorics)
  + [Modular RBP Molecular Modelling](https://2020.igem.org/Team%3AHeidelberg/Model/Pumilio_PPR)
* [Software](https://2020.igem.org/Team%3AHeidelberg/Software)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Software)
  + [PRISM](https://2020.igem.org/Team%3AHeidelberg/Software/PRISM)
  + [CoNCoRDE](https://2020.igem.org/Team%3AHeidelberg/Software/Concorde)
  + [3DOC](https://2020.igem.org/Team%3AHeidelberg/Software/3DOC)
  + [RISE](https://2020.igem.org/Team%3AHeidelberg/Software/RISE)
* [Human Practices](https://2020.igem.org/Team%3AHeidelberg/Human_Practices)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Human_Practices)
  + [Integrated Human Practices](https://2020.igem.org/Team%3AHeidelberg/Integrated)
  + [Expert Interviews](https://2020.igem.org/Team%3AHeidelberg/Interviews)
  + [Safety](https://2020.igem.org/Team%3AHeidelberg/Safety)
  + [Education](https://2020.igem.org/Team%3AHeidelberg/Education)
  + [Collaborations](https://2020.igem.org/Team%3AHeidelberg/Collaborations)
* [Notebook](https://2020.igem.org/Team%3AHeidelberg/Notebook)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Notebook)
  + [Methods](https://2020.igem.org/Team%3AHeidelberg/Experiments)
  + [Materials](https://2020.igem.org/Team%3AHeidelberg/Materials)
* [For Judges](https://2020.igem.org/Team%3AHeidelberg/Sitemap)

[The Legend of Cellda](https://2020.igem.org/Team%3AHeidelberg)

---

COLLAPSE NAVIGATION

---

* People
  + [Team](https://2020.igem.org/Team%3AHeidelberg/Team)
  + [Attributions](https://2020.igem.org/Team%3AHeidelberg/Attributions)
  + [Sponsors](https://2020.igem.org/Team%3AHeidelberg/Sponsoring)
* Project
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Description)
  + [RNA-Protein Interaction](https://2020.igem.org/Team%3AHeidelberg/RNA_Protein)
  + [PPR & Pumilio Domains](https://2020.igem.org/Team%3AHeidelberg/Pumilio_PPR)
  + [DNA:RNA Triple Helices](https://2020.igem.org/Team%3AHeidelberg/Triple_Helix)
  + [Split Trans Splicing Ribozymes](https://2020.igem.org/Team%3AHeidelberg/Trans_Splicing)
  + [Proposed Implementation](https://2020.igem.org/Team%3AHeidelberg/Implementation)
  + [Engineering Success](https://2020.igem.org/Team%3AHeidelberg/Engineering)
  + [Proof of Concept](https://2020.igem.org/Team%3AHeidelberg/Proof_Of_Concept)
  + [Contribution](https://2020.igem.org/Team%3AHeidelberg/Contribution)
  + [Achievements](https://2020.igem.org/Team%3AHeidelberg/Achievements)
* Parts
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Parts)
  + [RBP Collection](https://2020.igem.org/Team%3AHeidelberg/Part_Collection)
* Modeling
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Model)
  + [RNA Operads](https://2020.igem.org/Team%3AHeidelberg/Model/RNA_Combinatorics)
  + [Modular RBP Molecular Modelling](https://2020.igem.org/Team%3AHeidelberg/Model/Pumilio_PPR)
* Software
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Software)
  + [PRISM](https://2020.igem.org/Team%3AHeidelberg/Software/PRISM)
  + [CoNCoRDE](https://2020.igem.org/Team%3AHeidelberg/Software/Concorde)
  + [3DOC](https://2020.igem.org/Team%3AHeidelberg/Software/3DOC)
  + [RISE](https://2020.igem.org/Team%3AHeidelberg/Software/RISE)
* Human Practices
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Human_Practices)
  + [Integrated Human Practices](https://2020.igem.org/Team%3AHeidelberg/Integrated)
  + [Expert Interviews](https://2020.igem.org/Team%3AHeidelberg/Interviews)
  + [Safety](https://2020.igem.org/Team%3AHeidelberg/Safety)
  + [Education](https://2020.igem.org/Team%3AHeidelberg/Education)
  + [Collaborations](https://2020.igem.org/Team%3AHeidelberg/Collaborations)
  >
* Notebook
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Notebook)
  + [Methods](https://2020.igem.org/Team%3AHeidelberg/Experiments)
  + [Materials](https://2020.igem.org/Team%3AHeidelberg/Materials)
* [For Judges](https://2020.igem.org/Team%3AHeidelberg/Sitemap)

Engineering Success

Design, Build, Test, Learn...

# Introduction

iGEM is an engineering competition, about developing functional biological building blocks. The process of creating such building blocks is described by the iGEM engineering cycle, which starts with carefully designing a part, then building and testing it, learning from the results and finally improving the part with the gained knowledge. Because of Covid-19 it was quite challenging especially for our wetlab projects, to advance to the building and testing segment of the cycle. Nevertheless we made an effort to follow the principles, intensively researched literature and consulted experts to create well outlined plans for our experiments:

## Detailed plans of our work in the wetlab

![Card image cap](https://static.igem.org/mediawiki/2020/6/62/T--Heidelberg--iconrnaprotein.png)

#### RNA-Protein Interaction

In Protein-RNA interaction we developed design-principles for RNA binding proteins and RNA Linkers. Further we designed a Split-Reporter assay to be able to to measure the performance of these parts.

[RNA Protein Interaction](https://2020.igem.org/Team%3AHeidelberg/RNA_Protein)

![Card image cap](https://static.igem.org/mediawiki/2020/a/a1/T--Heidelberg--ppricon.png)

#### PPR and Pumby Proteins

PPR and Pumby proteins are useful RNA binding proteins which can be designed to bind any RNA sequences. We designed assays utilising them as RNA control elements and developed a part collection which allows the easy creation of novel PPR and Pumby proteins.

[PPR and Pumby Proteins](https://2020.igem.org/Team%3AHeidelberg/Pumilio_PPR)

![Card image cap](https://static.igem.org/mediawiki/2020/3/31/T--Heidelberg--rnaicon.png)

#### RNA-Triple Helix

We decided to explore the possibilities of using an RNA\*DNA-DNA triple helix transcriptional regulation. This tool could provide an improved alternative to the dCas protein.

[RNA-Triple-Helix](https://2020.igem.org/Team%3AHeidelberg/Triple_Helix)

![Card image cap](https://static.igem.org/mediawiki/2020/b/bf/T--Heidelberg--hymenaicon.png)

#### Split-Tetrahymena Ribozyme

In Split-Tetrahymena experiments we designed a highly modular version of this well known trans splicing ribozyme, and many different assays to evaluate the performance of the versions designed by us.

[Split-Tetrahymena Ribozyme](https://2020.igem.org/Team%3AHeidelberg/Trans_Splicing)

![Card image cap](https://static.igem.org/mediawiki/2020/7/78/T--Heidelberg--pipette.png)

#### Methods

The best experiment ideas are not of much use when one does not have the right protocols to support them. So, we assembled a variety of protocols that would have helped us in the experimental execution.

[Methods](https://2020.igem.org/Team%3AHeidelberg/Experiments)

## On unexpected results

The objective of science is to explore the unknown. Therefore it is impossible to predict all the possible results and solutions for them. Our strategy was to research carefully and use the available software tools like CONCORDE or the ViennaRNA webserver RNAfold, to increase the probability of our experiments functioning properly. In some cases we developed new software tools to address our problems. In case of unexpected errors we firstly planned to examine all the possible error sources in our experiments. We tried to design our experiments in the way that we could make sure that every element of the construct is working properly. If we wouldn’t be able to find the reason for an unexpected result, we would try to research the literature further or ask for advice from an expert.

## Examples for implemented engineering cycles in our experiments

### The Broccoli Aptamer

We wanted to create an RNA binding protein with endonuclease activity, since it would expand the abilities of cellular regulation on a transcriptional level. After rigorous research we decided to execute an experiment with a complex consisting of a Pentatricopeptide Repeat Protein (PPR) and a Ribonuclease A. The first problem we encountered while designing the experiment was that we could not use a mRNA translation inhibition assay like in the (more information on the design of mRNA translation inhibition), since the PPR could have an inhibitory effect on translation of the mRNA, the fluorescence loss could be contributed solely to the complexation of the PPR and mRNA and not necessarily to the degradation of the mRNA through the ribonuclease. We found the solution in using a broccoli aptamer, which is an RNA with stable secondary structure. Broccoli can bind the fluorogen DFHBI and therefore its presence can be detected by fluorescence. Because fluorescence is not induced through translation with the broccoli aptamer, we theorized that a loss of fluorescence would indeed only be associated with the degradation of the aptamer through the ribonuclease.
But beware, the use of Broccoli came with new challenges! Firstly, PPRs can bind only single stranded RNA, but Broccoli has a very stable secondary structure and has no suitable binding site for a PPR hexamer. Secondly, the original broccoli has a BsmbI cutting site, but we had to use BsmbI for the Marburg Collection cloning. These obstacles were resolved by redesigning the Broccoli aptamer computationally with a software we designed called [Designsimple](https://github.com/igemsoftware2020/Heidelberg_2020/blob/main/CoNcoRDe/rnasupport/scripts).
If we had enough time in the lab we would firstly try to collect evidence that the redesigned Broccoli aptamer functions. If that would not be the case, we would analyse what are the other possible ways to redesign the original Broccoli. Afterwards we would continue with our experiment.

### RNA\*DNA-DNA triple helices

RNA\*DNA-DNA triple helix have been known for a longer period of time. Recently, Kunkler et al. characterised binding energies of single RNA-DNA nucleotide pairs in the triple helix in vitro.triple\_helix\_1 They also showed that the minimal RNA length for binding is 19 base pairs. Based on this information we designed novel RNAs and DNAs that should theoretically fold into a triple helix. We planned on testing these constructs together with the sequence for which Kunkler et al. proved to form a triple helix in vitro. If the results would show that our constructs don’t fold into a triple helix, but the sequence proposed by Kunkler et al. does, we would try to increase the length of our constructs. If neither the sequence proposed by Kunkler et al. nor our constructs form a triple helix in E. coli, we would try to repeat the experiment in S. cerevisiae. This could address the problem, since it is predicted that the sequence proposed by Kunkler et al. forms a triple helix in human cells.

Another engineering question in the experiments with the triple helix was, which distance between the promoter and the binding site of the RNA should we choose. It was shown that an analogical construct using a dCas instead of a triple helix works optimally if the binding site of the guide RNA is 91 base pairs in front of the promoter.triple\_helix\_4 We compared the approximate size of our construct with the structure of dCas bound to the DNA.triple\_helix\_6 Based on the obtained information we decided to try designs with binding site 91, 71 and 61 base pairs in front of the promoter. We would compare the efficiency of activation and repression of those cases by measuring fluorescence. Our hypothesis is that the distance of 71 base pairs is optimal. If the results contradict the hypothesis we would try other distances from the promoter.

### PPR mRNA translation inhibition assay

The only measurement of our experiments that we were able to conduct, was the measurement of some of the plasmid constructs, designed for the mRNA translation inhibition assay (also see our  [PPR and Pumilio page](%E2%80%9D) for more information and results). In the measurement a positive control containing a sole sfGFP, a negative control containing a OFF Target PPR Protein and a sfGFP, and an ON Target PPR Protein and sfGFP were measured for their fluorescence intensity. In contrast to our expectation the ON target PPR Protein did not decrease the fluorescence of the sfGFP Reporter. Further, the OFF Target PPR seemed to decrease the fluorescence of the sfGFP greater than the ON Target PPR. In spite, of this disappointing outcome we would not directly abort this assay. The PPRs that were measured in the experiment, were expressed under the Anderson promoter BBa\_J23115, while the sfGFP reporter was expressed under the Anderson promoter BBa\_J23100, which is a promotor that that is >6 times stronger than BBa\_J23115. Additionally the PPR Proteins were cloned onto a plasmid containing a p15A ORI, while the sfGFP reporters where cloned into a plasmid with a ColE1 ORI, which has a higher copy number. In conclusion the reason for the bad result may simply be the overwhelming expression of sfGFP in the samples. If we would have had more time we would have conducted this assay again with other PPR constructs we designed, being that are expressed under stronger promoters, and potentially would try to clone the PPR constructs into a plasmid with another ORI which has a more similar copy number like ColE1.

### References

#### Quote

*“”*

#### Useful **Links**

[Special pages](https://igem.org/Special%3ASpecialPages)

[Main Page](https://igem.org/Main_Page)

#### Follow us on

[*Instagram*](https://www.instagram.com/igem.heidelberg/)
&
[*Twitter*](https://twitter.com/igemHD)

#### Contact us

igemhd
@protonmail.com

Im Neuenheimer Feld 267

69120 Heidelberg

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Heidelberg/Experiments

NAVIGATION

* [People](https://2020.igem.org/Team%3AHeidelberg/Team)
  + [Team](https://2020.igem.org/Team%3AHeidelberg/Team)
  + [Attributions](https://2020.igem.org/Team%3AHeidelberg/Attributions)
  + [Sponsors](https://2020.igem.org/Team%3AHeidelberg/Sponsoring)
* [Project](https://2020.igem.org/Team%3AHeidelberg/Description)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Description)
  + [RNA-Protein Interaction](https://2020.igem.org/Team%3AHeidelberg/RNA_Protein)
  + [PPR & Pumilio Domains](https://2020.igem.org/Team%3AHeidelberg/Pumilio_PPR)
  + [DNA:RNA Triple Helices](https://2020.igem.org/Team%3AHeidelberg/Triple_Helix)
  + [Split Trans Splicing Ribozymes](https://2020.igem.org/Team%3AHeidelberg/Trans_Splicing)
  + [Proposed Implementation](https://2020.igem.org/Team%3AHeidelberg/Implementation)
  + [Engineering Success](https://2020.igem.org/Team%3AHeidelberg/Engineering)
  + [Proof of Concept](https://2020.igem.org/Team%3AHeidelberg/Proof_Of_Concept)
  + [Contribution](https://2020.igem.org/Team%3AHeidelberg/Contribution)
  + [Achievements](https://2020.igem.org/Team%3AHeidelberg/Achievements)
* [Parts](https://2020.igem.org/Team%3AHeidelberg/Parts)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Parts)
  + [RBP Collection](https://2020.igem.org/Team%3AHeidelberg/Part_Collection)
* [Modeling](https://2020.igem.org/Team%3AHeidelberg/Model)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Model)
  + [RNA Operads](https://2020.igem.org/Team%3AHeidelberg/Model/RNA_Combinatorics)
  + [Modular RBP Molecular Modelling](https://2020.igem.org/Team%3AHeidelberg/Model/Pumilio_PPR)
* [Software](https://2020.igem.org/Team%3AHeidelberg/Software)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Software)
  + [PRISM](https://2020.igem.org/Team%3AHeidelberg/Software/PRISM)
  + [CoNCoRDE](https://2020.igem.org/Team%3AHeidelberg/Software/Concorde)
  + [3DOC](https://2020.igem.org/Team%3AHeidelberg/Software/3DOC)
  + [RISE](https://2020.igem.org/Team%3AHeidelberg/Software/RISE)
* [Human Practices](https://2020.igem.org/Team%3AHeidelberg/Human_Practices)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Human_Practices)
  + [Integrated Human Practices](https://2020.igem.org/Team%3AHeidelberg/Integrated)
  + [Expert Interviews](https://2020.igem.org/Team%3AHeidelberg/Interviews)
  + [Safety](https://2020.igem.org/Team%3AHeidelberg/Safety)
  + [Education](https://2020.igem.org/Team%3AHeidelberg/Education)
  + [Collaborations](https://2020.igem.org/Team%3AHeidelberg/Collaborations)
* [Notebook](https://2020.igem.org/Team%3AHeidelberg/Notebook)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Notebook)
  + [Methods](https://2020.igem.org/Team%3AHeidelberg/Experiments)
  + [Materials](https://2020.igem.org/Team%3AHeidelberg/Materials)
* [For Judges](https://2020.igem.org/Team%3AHeidelberg/Sitemap)

[The Legend of Cellda](https://2020.igem.org/Team%3AHeidelberg)

---

COLLAPSE NAVIGATION

---

* People
  + [Team](https://2020.igem.org/Team%3AHeidelberg/Team)
  + [Attributions](https://2020.igem.org/Team%3AHeidelberg/Attributions)
  + [Sponsors](https://2020.igem.org/Team%3AHeidelberg/Sponsoring)
* Project
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Description)
  + [RNA-Protein Interaction](https://2020.igem.org/Team%3AHeidelberg/RNA_Protein)
  + [PPR & Pumilio Domains](https://2020.igem.org/Team%3AHeidelberg/Pumilio_PPR)
  + [DNA:RNA Triple Helices](https://2020.igem.org/Team%3AHeidelberg/Triple_Helix)
  + [Split Trans Splicing Ribozymes](https://2020.igem.org/Team%3AHeidelberg/Trans_Splicing)
  + [Proposed Implementation](https://2020.igem.org/Team%3AHeidelberg/Implementation)
  + [Engineering Success](https://2020.igem.org/Team%3AHeidelberg/Engineering)
  + [Proof of Concept](https://2020.igem.org/Team%3AHeidelberg/Proof_Of_Concept)
  + [Contribution](https://2020.igem.org/Team%3AHeidelberg/Contribution)
  + [Achievements](https://2020.igem.org/Team%3AHeidelberg/Achievements)
* Parts
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Parts)
  + [RBP Collection](https://2020.igem.org/Team%3AHeidelberg/Part_Collection)
* Modeling
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Model)
  + [RNA Operads](https://2020.igem.org/Team%3AHeidelberg/Model/RNA_Combinatorics)
  + [Modular RBP Molecular Modelling](https://2020.igem.org/Team%3AHeidelberg/Model/Pumilio_PPR)
* Software
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Software)
  + [PRISM](https://2020.igem.org/Team%3AHeidelberg/Software/PRISM)
  + [CoNCoRDE](https://2020.igem.org/Team%3AHeidelberg/Software/Concorde)
  + [3DOC](https://2020.igem.org/Team%3AHeidelberg/Software/3DOC)
  + [RISE](https://2020.igem.org/Team%3AHeidelberg/Software/RISE)
* Human Practices
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Human_Practices)
  + [Integrated Human Practices](https://2020.igem.org/Team%3AHeidelberg/Integrated)
  + [Expert Interviews](https://2020.igem.org/Team%3AHeidelberg/Interviews)
  + [Safety](https://2020.igem.org/Team%3AHeidelberg/Safety)
  + [Education](https://2020.igem.org/Team%3AHeidelberg/Education)
  + [Collaborations](https://2020.igem.org/Team%3AHeidelberg/Collaborations)
  >
* Notebook
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Notebook)
  + [Methods](https://2020.igem.org/Team%3AHeidelberg/Experiments)
  + [Materials](https://2020.igem.org/Team%3AHeidelberg/Materials)
* [For Judges](https://2020.igem.org/Team%3AHeidelberg/Sitemap)

Methods

Me pipette? Yeah. Me contaminate? No.

#### [Agarose Gel Electrophoresis](#openbox-1-collapse)

Normally, gels were used with a concentration of 1 % agarose. However, for really small DNA fragments and for the gel extraction, gels with an agarose concentration of 2 % were used.
The following protocol was followed:
- The agarose was dissolved in 1x TAE buffer by boiling. Per 100 ml agarose solution 5 µl ethidium bromide was added.- The gel was poured into a gel electrophoresis chamber and it was waited until it completely cooled down. Then it was placed into the tank and it was filled up with 1x TAE buffer until the whole gel was covered.- 0.5 µl of a 6x purple loading dye were mixed with 2.5 µl of the sample. The mixtures were loaded into the gel pockets.- As a ladder, the DirectLoad Wide Range DNA Marker was used.- The gel was run at 90 V for around 40 minutes and imaged using a UV transilluminator.

#### [Calcium Competent Cells](#openbox-2-collapse)

The following buffers were prepared before beginning:
100 mM MgCl2, 100 mM CaCl2 and 85 mM CaCl2, 15 % glycerol
The buffers without glycerol were autoclaved. The buffer with glycerol was sterile filtered with a 0.22 µm filter.
The following protocol was followed to prepare the competent Top 10 cells:
- 10 ml LB medium was inoculated with the Top 10 strain (as a friendly gift of the AG Niopek) and incubated overnight at 37 °C and 225 rpm.- On the next day, two flasks of 500 ml LB medium were each inoculated with 5 ml of the overnight culture. It was grown to an OD600 of 0.35 - 0.4.- The cells were cooled for 30 minutes on ice. From this step on, the cells were kept on ice.- The cells were split into 20 pre-cooled 50 ml falcon tubes and centrifuged for 15 minutes at 3000 xg. The supernatant was discarded.- The pellet was resuspended in 20 ml of pre-cooled MgCl2. The mixture was centrifuged for 15 minutes at 2000 xg. Again, the supernatant was discarded.- The pellet was resuspended in 10 ml of pre-cooled CaCl2. The content of five falcon tubes was combined and chilled for 20 minutes on ice. Again, it was centrifuged for 15 minutes at 2000 xg.- The pellet was resuspended in 12.5 ml pre-cooled 85 mM CaCl2, 15 % glycerol. The content of the falcons was combined and centrifuged for 15 minutes at 2000 xg.- The pellet was resuspended in 2 ml pre-cooled 85 mM CaCl2, 15 % glycerol. Each 50 µl was aliquoted into pre-cooled Eppendorf tubes. The aliquots were snap-frozen in liquid nitrogen and stored at -80 °C.- Small amounts of the cells were spread on agar plates with resistance genes, to check whether the cells were contaminated with a resistance gene.

#### [Fluorescence Measurement with Broccoli](#openbox-21-collapse)

For the fluorescence measurement in the plate reader, the following protocol was followed:
- Competent Top 10 cells were transformed with Level 1 broccoli plasmids and grown on an agar plate.- Five colonies were picked per plate and an overnight seed culture prepared. Another seed culture is prepared with cells not transformed with a plasmid. This culture is referred to as crude culture.- On the next day, 3 ml of the seed cultures were diluted to an OD 600 of 0.4 in LB medium containing the appropriate antibiotics.- The dilution was incubated for two to four hours.- A buffer of 40 µM DFHBI in PBS was prepared. DFHBI was diluted beforehand in DMSO to a concentration of 40 mM.- 1 ml of the buffer was mixed with 20 µl of the seed culture and incubated for 30 minutes.- Two negative controls were prepared. One negative control was prepared with 20 µl LB medium and one with 20 µl of the crude culture, both mixed with 1 ml of the DFHBI-PBS buffer. The negative controls were also incubated for 30 minutes.- The OD600 of all cultures and negative controls was measured.- 100 µl of each culture and negative control were pipetted in wells of a black 96-well plate. Like this of each culture four replicates were made.- The fluorescence intensity was measured with a plate reader at an excitation maximum of 447 nm and an emission maximum of 501 nm.- For analysis the means of each replicate pairs were calculated. The background fluorescence (fluorescence intensities of PBS and 40 µM DFHBI medium without bacteria) were substracted. All fluorescence intensities were normalised to equal bacterial concentrations by dividing the values through the OD600 that was measured earlier. Finally, the mean values of the seed cultures of the broccoli colonies were divided by the mean value of the crude seed culture to compare the fluorescent intensity of the probes to the negative control.

#### [Measurement of fluorescent protein assays](#openbox-22-collapse)

For the measurement of fluorescent proteins, the following protocol was followed:
- Competent Top 10 cells were transformed with the appropriate plasmids to obtain colonies with the correct probes. The bacteria was grown on an agar plate overnight.- Five colonies were picked per plate and an overnight seed culture was prepared. Another seed culture is prepared with cells not transformed with a plasmid. This culture is referred to as crude culture.- On the next day, 3 ml of the seed cultures were diluted to an OD 600 of 0.4 in LB medium with the respective antibiotic.- It was incubated for two to four hours.- Two negative controls were prepared. One negative control was prepared with LB medium and one with the crude culture.- The OD of all cultures and negative controls was measured.- 100 µl of each culture and negative control were pipetted in wells of a black 96-well plate. Like this of each culture four replicates were made.- The fluorescence intensity was measured with a plate reader at an excitation maximum of 485 nm and an emission maximum of 510 nm in the case of sfGFP, at an excitation maximum of 468 nm and an emmision maximum of 505 nm in the case of assembled split-GFP or at an excitation maximum of 569 nm and emmission maximum of 594 nm in the case of mScarlet.- For analysis the means of each replicate pairs were calculated. The background fluorescence (fluorescence intensities of LB medium without bacteria) were substracted. All fluorescence intensities were normalised to equal bacterial concentrations by dividing the values through the OD600 that was measured earlier. Finally, the mean values of the probes were divided by the mean value of the crude seed culture, to compare the fluorescent intensities of the probes to the negative control

#### [Gel Extraction](#openbox-23-collapse)

Gel Extractions were performed with the NEB Monarch Gel Extraction Kit as proposed by the manufacturer. For gel extraction, a higher volume of the PCR reaction mixture was prepared. 100 µl of the PCR product was loaded on a gel. For getting big enough pockets in the gel, tape was clued around two tooths of a gel comb.

#### [Glycerol stocks](#openbox-24-collapse)

1 ml of an overnight culture were transferred in an Eppendorf tube and centrifuged at 2500 rpm for 15 minutes. The supernatant was decanted and the pellet was resuspended in 1 ml LB with 20 % glycerol. The glycerol stocks were sorted into freezer boxes and stored in the -80°C freezer.

#### [Golden Gate Cloning - Level 0](#openbox-3-collapse)

Level 0 Golden Gate Cloning was done with the following components:
- 75 ng PCR product (as the concentration could not be measured we used 2 µl)- 7.5 ng entry vector- 1 µl T4 DNA Ligase buffer- 1 µl T4 DNA Ligase or Hi-T4 DNA Ligase- 0.5 µl EspI3- Milli-Q water to 10 µl
In the thermocycler the following program was followed:

**Table 1: Thermocycler settings for Golden Gate Level 0 cloning.**
For the T4-Ligase the ligation temperature in Step 2 was changed to 16 °C.

Step |  || Step 1 | 37 °C, 1 min 30 sec |
| Step 2 | 25 °C, 3 min |
| Repetition of Step 1 and 2 | 15 x |
| Step 3 | 50 °C, 5 min |
| Step 4 | 80 °C, 10 min |
| Step 5 | 4 °C, until lid is opened |

After the cloning, an analytical gel electrophoresis was performed.

#### [Golden Gate Cloning - Level 1](#openbox-4-collapse)

Level 1 Golden Gate Cloning was done with the following components:
- 75 ng of each DNA insert- 1 µl T4 DNA Ligase buffer- 1 µl T4 DNA Ligase or Hi-T4 DNA Ligase- 0.5 µl Bsa3I- Milli-Q water to 10 µl
In the thermocycler the following program was followed:

**Table 2: Thermocycler settings for Golden Gate Level 1 cloning.**
For the T4-Ligase the ligation temperature in Step 2 was changed to 16 °C.

Step |  || Step 1 | 37 °C, 2 min |
| Step 2 | 25 °C, 5 min |
| Repetition of Step 1 and 2 | 50 x |
| Step 3 | 50 °C, 10 min |
| Step 4 | 80 °C, 10 min |
| Step 5 | 4 °C, until lid is opened |

Level 1 cloning was performed for plasmids gaining three different antibiotic resistances: Ampicillin-resistance, Kanamycin-resistance, Tetracycline-resistance.
After the cloning, an analytical gel electrophoresis was performed.

#### [Golden Gate Cloning - Level 2](#openbox-5-collapse)

Level 2 Golden Gate Cloning was done with the following components:
- 75 ng of each DNA insert- 1 µl T4 DNA Ligase buffer- 1 µl T4 DNA Ligase or Hi-T4 DNA Ligase- 0.5 µl EspI3- Milli-Q water to 10 µl
In the thermocycler the following program was followed:

**Table 3: Thermocycler settings for Golden Gate Level 2 cloning.**
For the T4-Ligase the ligation temperature in Step 3 was changed to 16 °C.

Step |  || Step 1 | 37 °C, 5 min |
| Step 2 | 25 °C, 16 min |
| Repetition of Step 1 and 2 | 30 x |
| Step 3 | 37 °C, 30 min |
| Step 4 | 80 °C, 10 min |
| Step 5 | 4 °C, until lid is opened |

**Note: We were not able to construct a finished Level 2 cloning construct in our limited time in the lab, but would highly suspect the thermocycler conditions stated in Table 3.**
After the cloning, an analytical gel electrophoresis was performed.

#### [Ligation](#openbox-51-collapse)

For example, the Pumbys had to be synthesized in two separate parts. To combine them, a digest with BsaI and a ligation were performed. The following components were combined, mixed by pipetting, and put in the thermocycler, following the program: incubate for 60 min (37 °C), deactivate 10 min (65 °C), stay at 4°C.
- 5 µl of Pumby C-terminal and N-terminal PCR products- 2.5 µl T4 DNA Ligase Buffer 10X- 1.2 µl T4 DNA Ligase (500 U)- 0.75 µl BsaI (15 U)- 10.55 µl Milli-Q water

#### [Minipreps (Promega Kit)](#openbox-52-collapse)

The minipreps were conducted with the PureYield Plasmid Miniprep System of Promega as proposed by the manufacturer. The alternative protocol for larger culture volumes was amended. At first, 1.5 ml of bacterial culture were centrifuged at 10 000 rpm for 30 seconds. The supernatant was discarded and another 900 µl of the bacteria culture was added to the pellet and also centrifuged. The now resulting pellet was resuspended in another 600 µl bacterial culture.
After the miniprep, the concentration was measured with the Implen N80 Nanophotometer. As a blank, the elution buffer was used.

#### [Overlap Extension PCR](#openbox-53-collapse)

Overlap extension PCRs were performed with double the amount of primer volume.
The following was combined into a PCR-Tube:
- 0.5 µl of each primer at a concentration of 50 µM- 1 µl T4 DNA Ligase buffer- 10 ng DNA template- 12.5 µl PCR Mastermix (NEBNext® High-Fidelity 2X PCR Master Mix or NEB Taq 2X Master Mix)- Milli-Q water to 25 µl
In the thermocycler, the following program was used for the overlap extension PCRs:

**Table 4: Thermocycler settings for PCRs.**
The annealing temperature (Step 3) and extension time (Step 4) were determined for each primer pair with the NEB Tm calculator. For the used temperatures and times, check out our notebook.

Step | High-Fidelity 2X PCR Master Mix | Taq 2X Master Mix || Step 1 (Initial Denaturation) | 98 °C, 30 sec | 95 °C, 30 sec |
| Step 2 (Denaturation) | 98 °C, 10 sec | 95 °C, 25 sec |
| Step 3 (Annealing) | **variable**, 20 sec | **variable**, 20 sec |
| Step 4 (Extension) | 72 °C, **variable** | 68 °C, **variable** |
| Repetition of Steps 2 to 4 | 30 x | 30 x |
| Step 5 (Last Extension) | 72 °C, 2 min | 68 °C, 2 min |
| Step 6 | 4 °C, until lid is opened | 4 °C, until lid is opened |

The PCRs were verified with analytical Gel electrophoresis.

#### [PCR](#openbox-54-collapse)

PCRs were performed with the DNA parts and primers from IDT. The following volumes were combined in a PCR tube:
- 0.25 µl of each primer (Reverse and Forward) at a concentration of 50 µM- 1 µl T4 DNA Ligase buffer- 10 ng DNA template- 12.5 µl PCR Mastermix (NEBNext® High-Fidelity 2X PCR Master Mix or NEB Taq 2X Master Mix)- Milli-Q water to 25 µl
In the thermocycler, the following program was used for the PCRs:

**Table 5: Thermocycler settings for PCRs.**
The annealing temperature (Step 3) and extension time (Step 4) were determined for each primer pair with the NEB Tm calculator. For the used temperatures and times, check out our notebook.

Step | High-Fidelity 2X PCR Master Mix | Taq 2X Master Mix || Step 1 (Initial Denaturation) | 98 °C, 30 sec | 95 °C, 30 sec |
| Step 2 (Denaturation) | 98 °C, 10 sec | 95 °C, 25 sec |
| Step 3 (Annealing) | **variable**, 20 sec | **variable**, 20 sec |
| Step 4 (Extension) | 72 °C, **variable** | 68 °C, **variable** |
| Repetition of Steps 2 to 4 | 30 x | 30 x |
| Step 5 (Last Extension) | 72 °C, 2 min | 68 °C, 2 min |
| Step 6 | 4 °C, until lid is opened | 4 °C, until lid is opened |

#### [Picking of colonies and Colony PCR](#openbox-6-collapse)

Master mixes were prepared from water and the primers for the colony PCR. 490 µl water was mixed with 5 µl of both primers. Different primers were used for the Level 0 cloning, the Level 1 cloning with the various antibiotic resistances, and the Level 2 cloning. The primers were beforehand diluted to a concentration of 50 µM.
Before picking, Eppendorf tubes were prepared with 15 µl of the respective master mix. The colonies were picked with a small pipette tip. The pipette tip was first dipped two to three times in the prepared Eppendorf tube. Then, the pipette tip was thrown in a falcon for the overnight cultures (see Picking of colonies and overnight cultures).
Normally and if possible, five to ten colonies were picked per plate for bacteria transformed with Level 1 and Level 2 cloning constructs. Around three colonies were picked for bacteria transformed with Level 0 cloning constructs. It was determined with a UV transilluminator, which colonies were picked. White colonies were picked that did not emit a GFP signal.
To the master mix with cells, 12.5 µl PCR Mastermix (NEBNext® High-Fidelity 2X PCR Master Mix or NEB Taq 2X Master Mix) was added.
In the thermocycler, the following program was used for the colony PCR:

**Table 6: Thermocycler settings for colony PCRs.**
The annealing temperature (Step 3) depends on the used primers. For the Level 0 primers it is 53 °C, for the Level 1 ampicillin primers 46 °C and the tetracycline primers 51 °C and for the Level 2 primers 50 °C. For kanamycin-resistance genes no primers were available.

Step | High-Fidelity 2X PCR Master Mix | Taq 2X Master Mix || Step 1 (Initial Denaturation) | 98 °C, 30 sec | 95 °C, 30 sec |
| Step 2 (Denaturation) | 98 °C, 10 sec | 95 °C, 25 sec |
| Step 3 (Annealing) | **variable**, 30 sec | **variable**, 30 sec |
| Step 4 (Extension) | 72 °C, 2 min | 68 °C, 2 min |
| Repetition of Steps 2 to 4 | 30 x | 30 x |
| Step 5 (Last Extension) | 72 °C, 5 min | 68 °C, 5 min |
| Step 6 | 4 °C, until lid is opened | 4 °C, until lid is opened |

After the cloning, an analytical gel electrophoresis was performed.

#### [Picking of colonies and overnight cultures](#openbox-7-collapse)

A falcon was prepared with 4 ml LB medium inoculated with 4 µl of the preferred antibiotic. The antibiotics were used in the following concentrations: Ampicillin 100 µg/ml, Chloramphenicol 34 µg/ml, Kanamycin 30 µg/ml, Tetracycline 5 µg/ml.
The colonies were picked with a small pipette tip. The pipette tip was first dipped two to three times in an Eppendorf tube for the Colony PCR (see Picking of colonies and Colony PCR). Then, the pipette tip was thrown in the prepared falcon. The falcon was incubated at 37 °C and 225 rpm overnight.
Normally and if possible, five to ten colonies were picked per plate for bacteria transformed with Level 1 and Level 2 cloning constructs. Around three colonies were picked for bacteria transformed with Level 0 cloning constructs. It was determined with a UV transilluminator, which colonies were picked. White colonies were picked that did not emit a GFP signal.

#### [Pouring of agarplates](#openbox-71-collapse)

The autoclaved LB agar was heated in the microwave until it was a clear liquid. It then was cooled until its temperature was approximately 55°C. The preferred antibiotics were added in the following concentrations: Ampicillin 100 µg/ml, Chloramphenicol 34 µg/ml, Kanamycin 30 µg/ml, Tetracycline 5 µg/ml.
If two antibiotics were combined, the same concentrations were used.

#### [Preparation of antibiotics stocks](#openbox-8-collapse)

The antibiotics were prepared in the following dilutions: Ampicillin 100 mg/ml, Chloramphenicol 34 mg/ml, Kanamycin 30 mg/ml, Tetracycline 5 mg/ml.
Ampicillin and Kanamycin were diluted in Milli-Q water and were filter sterilized. Chloramphenicol and Tetracycline were diluted in 99.8 % ethanol.

#### [Preparation of LB medium and agar](#openbox-9-collapse)

For the LB liquid medium, 1 % Bacto-Tryptone, 0.5 % Bacto-Yeast Extract, and 0.17 M NaCl were mixed and autoclaved.
For the LB agar, the LB medium was mixed with 1.5 % agar and autoclaved. The LB agar was heated until it was a clear liquid. It was waited until the liquid cooled down to around 55 °C. The preferred antibiotics were added in the following concentrations: Ampicillin 100 µg/ml, Chloramphenicol 34 µg/ml, Kanamycin 30 µg/ml, Tetracycline 5 µg/ml.

#### [Preparation of SOC Medium](#openbox-10-collapse)

First SOB (Super Optimal Broth) Medium was prepared from 2 % Bacto-Tryptone, 0.5 % Bacto-Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and autoclaved.
100 ml of the autoclaved SOB medium were mixed with a filter-sterilized 20 % glucose solution. The glucose solution was filtered with a 0.22 µm filter.

#### [Resuspension of DNA and primers from IDT](#openbox-101-collapse)

The DNA parts and primers which were synthesized by IDT were shipped dry. They were resuspended in the following way: The tubes were centrifuged for 5 s, Milli-Q water was added to reach a concentration of 10 ng/µl and the tubes were vortexed briefly. Afterward, they were incubated 15 minutes at 50 °C, vortexed briefly, and spun down. The resuspended DNA was stored in the -20 °C freezer.
The primers were resuspended in the following way: The tubes were centrifuged for 5 s, Milli-Q water was added to reach a 50µM solution (20 times the oligo yield in nmol) and the tubes were vortexed briefly and spun down. The resuspended DNA was stored in the -20 °C freezer.

#### [Sanger Sequencing](#openbox-102-collapse)

From the Minipreps we prepared, we took samples and prepared them for sequencing in Economy Sanger Sequences from Microsynth. The probes were prepared after the directions of Microsynth. Tubes with a total volume of 15 µl were prepared with 3 µl 20 µM Sequencing Primer (1.2 µl 50 µM Primer), and the appropriate amounts of nuclease-free water and plasmid based on the initial concentration of the prepped plasmids. The plasmids were diluted in water to 60 - 80 ng per 15 µl.

#### [Transformation](#openbox-11-collapse)

Transformations were performed with the method of heat shock. Competent Top 10 cells were, still frozen, inoculated with 10 µl of the desired plasmid, and incubated for 30 minutes on ice. The heat shock was performed for 30 seconds at 42 °C. The cells were incubated for another 2 minutes on ice. 1 ml of SOC medium was added. The cells in the SOC medium were incubated at 37 °C and 300 rpm. Incubation time depended on the used antibiotic. For ampicillin, the incubation time was one hour, for chloramphenicol, kanamycin, and tetracycline the incubation time was two hours. 100 to 300 µl of the cells were spread on an agar plate with the appropriate antibiotic. The plates were incubated overnight at 37 °C.
The protocol of Molecular and Cell Biology, Berkely was followed.

#### Quote

*“”*

#### Useful **Links**

[Special pages](https://igem.org/Special%3ASpecialPages)

[Main Page](https://igem.org/Main_Page)

#### Follow us on

[*Instagram*](https://www.instagram.com/igem.heidelberg/)
&
[*Twitter*](https://twitter.com/igemHD)

#### Contact us

igemhd
@protonmail.com

Im Neuenheimer Feld 267

69120 Heidelberg

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Heidelberg/Results

NAVIGATION

* [People](https://2020.igem.org/Team%3AHeidelberg/Team)
  + [Team](https://2020.igem.org/Team%3AHeidelberg/Team)
  + [Attributions](https://2020.igem.org/Team%3AHeidelberg/Attributions)
  + [Sponsors](https://2020.igem.org/Team%3AHeidelberg/Sponsoring)
* [Project](https://2020.igem.org/Team%3AHeidelberg/Description)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Description)
  + [RNA-Protein Interaction](https://2020.igem.org/Team%3AHeidelberg/RNA_Protein)
  + [PPR & Pumilio Domains](https://2020.igem.org/Team%3AHeidelberg/Pumilio_PPR)
  + [DNA:RNA Triple Helices](https://2020.igem.org/Team%3AHeidelberg/Triple_Helix)
  + [Split Trans Splicing Ribozymes](https://2020.igem.org/Team%3AHeidelberg/Trans_Splicing)
  + [Proposed Implementation](https://2020.igem.org/Team%3AHeidelberg/Implementation)
  + [Engineering Success](https://2020.igem.org/Team%3AHeidelberg/Engineering)
  + [Proof of Concept](https://2020.igem.org/Team%3AHeidelberg/Proof_Of_Concept)
  + [Contribution](https://2020.igem.org/Team%3AHeidelberg/Contribution)
  + [Achievements](https://2020.igem.org/Team%3AHeidelberg/Achievements)
* [Parts](https://2020.igem.org/Team%3AHeidelberg/Parts)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Parts)
  + [RBP Collection](https://2020.igem.org/Team%3AHeidelberg/Part_Collection)
* [Modeling](https://2020.igem.org/Team%3AHeidelberg/Model)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Model)
  + [RNA Operads](https://2020.igem.org/Team%3AHeidelberg/Model/RNA_Combinatorics)
  + [Modular RBP Molecular Modelling](https://2020.igem.org/Team%3AHeidelberg/Model/Pumilio_PPR)
* [Software](https://2020.igem.org/Team%3AHeidelberg/Software)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Software)
  + [PRISM](https://2020.igem.org/Team%3AHeidelberg/Software/PRISM)
  + [CoNCoRDE](https://2020.igem.org/Team%3AHeidelberg/Software/Concorde)
  + [3DOC](https://2020.igem.org/Team%3AHeidelberg/Software/3DOC)
  + [RISE](https://2020.igem.org/Team%3AHeidelberg/Software/RISE)
* [Human Practices](https://2020.igem.org/Team%3AHeidelberg/Human_Practices)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Human_Practices)
  + [Integrated Human Practices](https://2020.igem.org/Team%3AHeidelberg/Integrated)
  + [Expert Interviews](https://2020.igem.org/Team%3AHeidelberg/Interviews)
  + [Safety](https://2020.igem.org/Team%3AHeidelberg/Safety)
  + [Education](https://2020.igem.org/Team%3AHeidelberg/Education)
  + [Collaborations](https://2020.igem.org/Team%3AHeidelberg/Collaborations)
* [Notebook](https://2020.igem.org/Team%3AHeidelberg/Notebook)
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Notebook)
  + [Methods](https://2020.igem.org/Team%3AHeidelberg/Experiments)
  + [Materials](https://2020.igem.org/Team%3AHeidelberg/Materials)
* [For Judges](https://2020.igem.org/Team%3AHeidelberg/Sitemap)

[The Legend of Cellda](https://2020.igem.org/Team%3AHeidelberg)

---

COLLAPSE NAVIGATION

---

* People
  + [Team](https://2020.igem.org/Team%3AHeidelberg/Team)
  + [Attributions](https://2020.igem.org/Team%3AHeidelberg/Attributions)
  + [Sponsors](https://2020.igem.org/Team%3AHeidelberg/Sponsoring)
* Project
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Description)
  + [RNA-Protein Interaction](https://2020.igem.org/Team%3AHeidelberg/RNA_Protein)
  + [PPR & Pumilio Domains](https://2020.igem.org/Team%3AHeidelberg/Pumilio_PPR)
  + [DNA:RNA Triple Helices](https://2020.igem.org/Team%3AHeidelberg/Triple_Helix)
  + [Split Trans Splicing Ribozymes](https://2020.igem.org/Team%3AHeidelberg/Trans_Splicing)
  + [Proposed Implementation](https://2020.igem.org/Team%3AHeidelberg/Implementation)
  + [Engineering Success](https://2020.igem.org/Team%3AHeidelberg/Engineering)
  + [Proof of Concept](https://2020.igem.org/Team%3AHeidelberg/Proof_Of_Concept)
  + [Contribution](https://2020.igem.org/Team%3AHeidelberg/Contribution)
  + [Achievements](https://2020.igem.org/Team%3AHeidelberg/Achievements)
* Parts
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Parts)
  + [RBP Collection](https://2020.igem.org/Team%3AHeidelberg/Part_Collection)
* Modeling
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Model)
  + [RNA Operads](https://2020.igem.org/Team%3AHeidelberg/Model/RNA_Combinatorics)
  + [Modular RBP Molecular Modelling](https://2020.igem.org/Team%3AHeidelberg/Model/Pumilio_PPR)
* Software
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Software)
  + [PRISM](https://2020.igem.org/Team%3AHeidelberg/Software/PRISM)
  + [CoNCoRDE](https://2020.igem.org/Team%3AHeidelberg/Software/Concorde)
  + [3DOC](https://2020.igem.org/Team%3AHeidelberg/Software/3DOC)
  + [RISE](https://2020.igem.org/Team%3AHeidelberg/Software/RISE)
* Human Practices
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Human_Practices)
  + [Integrated Human Practices](https://2020.igem.org/Team%3AHeidelberg/Integrated)
  + [Expert Interviews](https://2020.igem.org/Team%3AHeidelberg/Interviews)
  + [Safety](https://2020.igem.org/Team%3AHeidelberg/Safety)
  + [Education](https://2020.igem.org/Team%3AHeidelberg/Education)
  + [Collaborations](https://2020.igem.org/Team%3AHeidelberg/Collaborations)
  >
* Notebook
  + [Overview](https://2020.igem.org/Team%3AHeidelberg/Notebook)
  + [Methods](https://2020.igem.org/Team%3AHeidelberg/Experiments)
  + [Materials](https://2020.igem.org/Team%3AHeidelberg/Materials)
* [For Judges](https://2020.igem.org/Team%3AHeidelberg/Sitemap)

Results

Quo vadis wetlab?

# Introduction

PRISM -

PRISM (**P**rotein **R**NA **I**nteraction **S**equence **M**odeling) is a Language Model. It generates novel RNA-binding proteins, based on a user-inputted RNA-motif and characteristics of the protein, which differ significantly from known RNA-binding proteins.

3DOC -

3DOC (**3**D **do**main **c**oncatenation) generates PDBs (3D-Models) by concatenating modular protein domains or fusion protein parts and models them via PyRosetta and trRosetta. 3DOC and the provided pipelines makes modeling Protein-RNA complexes via Rosetta as easy as never before.

CoNcoRDe -

RISE -

RISE (iGEM **R**egistry **i**ntelligent **s**earch **e**ngine) provides an update for the iGEM Registry.The iGEM registry is a valuable resource for scientists. Adding functionality to filter entries and export sequences in various formats makes it an easy-to-use tool for future iGEM generations.

### References

#### Quote

*“”*

#### Useful **Links**

[Special pages](https://igem.org/Special%3ASpecialPages)

[Main Page](https://igem.org/Main_Page)

#### Follow us on

[*Instagram*](https://www.instagram.com/igem.heidelberg/)
&
[*Twitter*](https://twitter.com/igemHD)

#### Contact us

igemhd
@protonmail.com

Im Neuenheimer Feld 267

69120 Heidelberg