iGEM 2021 — Team TU_Kaiserslautern

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# Team:TU Kaiserslautern/Notebook

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[MOCLO MANIA](https://2021.igem.org/Team%3ATU_Kaiserslautern)

* [Home](https://2021.igem.org/Team%3ATU_Kaiserslautern)
* Team
  + [Team](https://2021.igem.org/Team%3ATU_Kaiserslautern/Team)
  + [Attributions](https://2021.igem.org/Team%3ATU_Kaiserslautern/Attributions)
* Project
  + [Description](https://2021.igem.org/Team%3ATU_Kaiserslautern/Description)
  + [Implementation](https://2021.igem.org/Team%3ATU_Kaiserslautern/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3ATU_Kaiserslautern/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3ATU_Kaiserslautern/Results)
  + [Notebook](https://2021.igem.org/Team%3ATU_Kaiserslautern/Notebook)
  + [Model](https://2021.igem.org/Team%3ATU_Kaiserslautern/Model)
* Laboratory
  + [Engineering](https://2021.igem.org/Team%3ATU_Kaiserslautern/Engineering)
  + [Contribution](https://2021.igem.org/Team%3ATU_Kaiserslautern/Contribution)
  + [Experiments](https://2021.igem.org/Team%3ATU_Kaiserslautern/Experiments)
  + [Safety](https://2021.igem.org/Team%3ATU_Kaiserslautern/Safety)
* Part Collection
  + [MoClo Mania Collection](https://2021.igem.org/Team%3ATU_Kaiserslautern/Parts)
  + [Composite Parts](https://2021.igem.org/Team%3ATU_Kaiserslautern/Composite_Parts)
* How To
  + [MoClo](https://2021.igem.org/Team%3ATU_Kaiserslautern/HowToMoClo)
  + [Leishmania](https://2021.igem.org/Team%3ATU_Kaiserslautern/HowToLeishmania)
* Human Practices
  + [Human Practices](https://2021.igem.org/Team%3ATU_Kaiserslautern/Human_Practices)
  + [Education and Communication](https://2021.igem.org/Team%3ATU_Kaiserslautern/Education)
  + [Collaborations](https://2021.igem.org/Team%3ATU_Kaiserslautern/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ATU_Kaiserslautern/Partnership)
* [Sponsors](https://2021.igem.org/Team%3ATU_Kaiserslautern/Sponsors)
* [Awards](https://2021.igem.org/Team%3ATU_Kaiserslautern/Awards)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3781)

[September '20](#head-1)
[October '20](#head-2)
[November '20](#head-3)
[December '20](#head-4)
[February '21](#head-5)
[March '21](#head-6)
[April '21](#head-7)
[May '21](#head-8)
[June '21](#head-9)
[July '21](#head-10)
[August '21](#head-11)
[September '21](#head-12)
[October '21](#head-13)
[November '21](#head-14)

# Notebook

## September '20

| When | What | Who |
| --- | --- | --- |
| 22.09.20 | First information evening and meeting | 2019/2020 Team + interested people |

## October '20

| When | What | Who |
| --- | --- | --- |
| 15.10.20 | Second information evening | 2019/2020 Team + interested people |
| 26.10.20 | First brainstorming | 2019/2020 Team + interested people |

## November '20

| When | What | Who |
| --- | --- | --- |
| 23.11.20 | Second brainstorming | Michael, Justus, Anna, Doro, Adrian + Team |
| 30.11.20 | Another Brainstorming | Team + Michael, Justus, Anna, Doro, Adrian, Marcel, Nora, Christina |

## December '20

| When | What | Who |
| --- | --- | --- |
| 07.12.20 | Meeting on how to structure an iGEM Team | Doro, Adrian + Team |
| 15.12.20 | Evaluating the remaining ideas with PIs and instructors | Michael, Justus, Anna, Nora, Nicole, Marcel, Doro, Adrian |
| 17.12.20 | Decision on project | Team |
| 21.12.20 | Introduction into sponsoring | Sponsoring team + advisors |

## February '21

| When | What | Who |
| --- | --- | --- |
| 01.02.21 | Starting with regular meetings on Mondays | Team + Doro & Adrian |
| 15.02.21 | First lab day: starting with domestication of plasmid – PCR | Jonas, Salina, Nicolas, Justus, Anna |

## March '21

| When | What | Who |
| --- | --- | --- |
| 05.03.21 | First Meeting with Collaboration Partner iGEM Bonn | Alex, Annika, Julia, Bastian |
| 09.03.21 | First L0-construct | Lab – Nicolas, Jonas, Salina |
| 12.03.21 | Domesticated Vector sequencing: missing 150 bp | MoClo Lab |
| 15.03.21 | Meeting our first Sponsor - Labfolder | Jonas, Tabea, Julia |
| 29.03.21 | Release of own [website](https://www.uni-kl.de/hochschulgruppen/iGEM/) |  |
| 25.03.21 | Meetup with Bonn | Julia, Bastian, Annika, Alex, Paul |
| 26.03.21 | Meeting GenScript Sponsoring | Julia, Salina, Bastian |
| 26.03.21 | Ohio State Meetup international | Tabea, Salina, Julia |
| 29.03.21 | Meetup Carl Roth sponsoring | Julia, Tabea, Benjamin |

## April '21

| When | What | Who |
| --- | --- | --- |
| 15.04.21 | Meeting Bonn – Science Slam & German Meetup | Alex, Julia, Annika, Bastian |
| 26.04.21 | First episode of our [Podcast "Genomgenommen"](https://open.spotify.com/show/1fjGmncU0Y0qTjDYDXefU5?si=c1cda377aabf4615) | Benjamin, Nicolas |
| 28.04.21 | Meeting Heidolph Instruments | Julia, Tabea, Benjamin |
| 28.04.21 | First L1 MoClo L1\_SAP\_RBD\_Strep8His & L1\_3xHA\_RBD\_Strep8His |  |
| 29.04.21 | Meeting Bonn | Julia, Bastian, Alex, Annika |

## May '21

| When | What | Who |
| --- | --- | --- |
| 03.05.21 | First test digest of L1\_3xHA\_RBD\_Strep8His (looking good) |  |
| 04.05.21. | Meeting Bonn Science Slam | Alex, Annika, Julia, Bastian |
| 05.05.21 | Meeting Merck | Tabea, Paul |
| 07.05.21 | Meeting Bonn German Meetup | Annika, Tabea, Julia, Alex |
| 11.05.21 | First transfection of *Leishmania* | Benjamin, Katrin, Tabea + Luzia |
| 12.05.21 | Escape Room Meeting: Bonn/KL/Düsseldorf/Aachen | Tabea, Jonas, Paul |
| 14.05.21 | [German Meetup](https://2021.igem.org/Team%3ATU_Kaiserslautern/Education#head-2) - Bonn & Kaiserslautern | Members from both teams + guests |
| 15.05.21 | Opening weekend festival | Team |
| 16.05.21 | Introduction stream | Team |
| 20.05.21 | [Blog entry Eurofins](https://the-dna-universe.com/2021/05/20/revolutionising-human-like-protein-production/) | Julia, Nicolas |
| 24.05.21 | First WIKI meeting | Team |
| 26.05.21 | First induction of *leishmania* with tetracycline | Benjamin, Katrin, Tabea + Luzia |

## June '21

| When | What | Who |
| --- | --- | --- |
| 04.06.21 | Meeting Bonn + Test Stream with Slammers | Slammers, Alex, Annika, Julia |
| 08.06.21 | Meeting Justin Mirus Vectorbuilder | Nicolas, Julia, Justin, Justus, Anna Kiefer |
| 09.06.21 | Presentation at Junior University | 65 kids, Bastian, Julia |
| 15.06.21 | School presentation – Bruchsal, St. Paulusheim | Salina, Julia, Katrin |
| 15.06.21 | Meeting Bonn, Moderation Slam | Julia, Alex |
| 18.06.21 | Science Slam | Team + 180 viewers + Slammers |
| 25.06.21 | Teambuilding Bonn | Kaiserslautern + Bonn |

## July '21

| When | What | Who |
| --- | --- | --- |
| 06.07.21 | School presentation - Saarbrücken, Deutsch-Französisches Gymnasium | Salina, Tabea, Benjamin |
| 07.07.21 | School presentation - Kaiserslautern, Albert-Schweizer-Gymnasium | Katrin, Tabea |
| 08.07.21 | Escape Room Meeting | Aachen, Düsseldorf, Bonn, Kaiserslautern |
| 12.07.21 | Meetup Jena Bioscience Dr. Licht | Team + Michael, Marcel, Anna, Justus, Luzia |
| 15.07.21 | Final domesticated vector - no base pairs missing |  || 22.07.21 | [Promotion Video](https://video.igem.org/w/5FyZZbR16rEGZTqUXaZZyM) | Team |

## August '21

| When | What | Who |
| --- | --- | --- |
| 02.08.21 | Fluorescence detection of colonies | Katrin, Tabea, Benjamin, Anna |
| 02.08.21 | Test run protein purification | Nicolas, Julia |
| 07.08.21 | Team Building | Team + Justus + Luzia |
| 12.08.21 | Fluorescence spectrum of mCherry and different cultures | Katrin, Tabea, Benjamin |
| 15.08.21 | Maastricht Vector–journal deadline | Nicolas, Julia |
| 11.08.21 | Interview with [Unispectrum live](https://www.unispectrum.de/forschen/kaiserslauterer-igem-team-nutzt-leishmanien-als-proteinproduzenten) live | Benjamin, Julia |
| 23.08.21 | Starting activity assay with ACE2 and RBD | Salina + Anna Kiefer |
| 27.08.21 | Meeting Justin Mirus | Justin, Nicolas, Julia, Justus |
| 27.08.21 | Meeting Andreas Licht | Team + Andreas Licht + Michael, Justus, Anna |
| 31.08.21 | Escape Room Meeting | Aachen, Bonn, Düsseldorf |

## September '21

| When | What | Who |
| --- | --- | --- |
| 03.09.21 | STEM fair – project presentation | Julia, Salina, Tabea, Bastian |
| 04.09.21 | STEM fair: Biology lab for kids | Katrin, Nicolas, Jonas, Benjamin |
| 07.09.21 | Vectorbuilder Special Seminar | Team + Justin Mirus + colleagues |
| 11.09.21 | Paris MeetUp digital | Team |
| 13.09.21 | First Western Blot showing RBD | Tabea, Benjamin, Katrin |

## October '21

| When | What | Who |
| --- | --- | --- |
| 04.10.21 | Recording podcast with iGEM Nawi Graz | Benjamin, Nicolas, Daniela |
| 07.10.21 | Meeting with Christian Janzen | Team + Christian Janzen + PhD Student |
| 18.10.21 | Meeting with Matthias Mieves | Salina, Julia |
| 21.10.21 | Wiki freeze | Team |
| 27.10.21 | Presentation video | Team |

## November '21

| When | What | Who |
| --- | --- | --- |
| 05.11.21 | Judging session | Team |

[![](https://static.igem.org/mediawiki/2021/d/dd/T--TU_Kaiserslautern--LogoImplen.jpg)](https://2021.igem.org/Team%3ATU_Kaiserslautern/Sponsors#implen)

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iGEM Kaiserslautern

**2021**

*made by [jules](https://stonedstreet.de)*

[Website](https://www.uni-kl.de/hochschulgruppen/iGEM/en/hello/)
Facebook
[Twitter](https://twitter.com/igem_tukl)
[Instagram](https://www.instagram.com/igem.kaiserslautern)
igem@bio.uni-kl.de

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# Team:TU Kaiserslautern/Engineering

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[MOCLO MANIA](https://2021.igem.org/Team%3ATU_Kaiserslautern)

* [Home](https://2021.igem.org/Team%3ATU_Kaiserslautern)
* Team
  + [Team](https://2021.igem.org/Team%3ATU_Kaiserslautern/Team)
  + [Attributions](https://2021.igem.org/Team%3ATU_Kaiserslautern/Attributions)
* Project
  + [Description](https://2021.igem.org/Team%3ATU_Kaiserslautern/Description)
  + [Implementation](https://2021.igem.org/Team%3ATU_Kaiserslautern/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3ATU_Kaiserslautern/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3ATU_Kaiserslautern/Results)
  + [Notebook](https://2021.igem.org/Team%3ATU_Kaiserslautern/Notebook)
  + [Model](https://2021.igem.org/Team%3ATU_Kaiserslautern/Model)
* Laboratory
  + [Engineering](https://2021.igem.org/Team%3ATU_Kaiserslautern/Engineering)
  + [Contribution](https://2021.igem.org/Team%3ATU_Kaiserslautern/Contribution)
  + [Experiments](https://2021.igem.org/Team%3ATU_Kaiserslautern/Experiments)
  + [Safety](https://2021.igem.org/Team%3ATU_Kaiserslautern/Safety)
* Part Collection
  + [MoClo Mania Collection](https://2021.igem.org/Team%3ATU_Kaiserslautern/Parts)
  + [Composite Parts](https://2021.igem.org/Team%3ATU_Kaiserslautern/Composite_Parts)
* How To
  + [MoClo](https://2021.igem.org/Team%3ATU_Kaiserslautern/HowToMoClo)
  + [Leishmania](https://2021.igem.org/Team%3ATU_Kaiserslautern/HowToLeishmania)
* Human Practices
  + [Human Practices](https://2021.igem.org/Team%3ATU_Kaiserslautern/Human_Practices)
  + [Education and Communication](https://2021.igem.org/Team%3ATU_Kaiserslautern/Education)
  + [Collaborations](https://2021.igem.org/Team%3ATU_Kaiserslautern/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ATU_Kaiserslautern/Partnership)
* [Sponsors](https://2021.igem.org/Team%3ATU_Kaiserslautern/Sponsors)
* [Awards](https://2021.igem.org/Team%3ATU_Kaiserslautern/Awards)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3781)

[Design](#head-1)
[Construction](#head-2)
[Expression](#head-3)
[Purification](#head-4)

# ENGINEERING SUCCESS

Since our project revolved around the **creation of a **genetic parts library****, the design, cloning and testing of basic parts was our daily business. Depending on the genetic source of the respective part, the design process either aimed to **create a sequence** ready for commercial **synthesis** or to generate a **set of primers** that would allow for **amplification** of the desired sequence from a **source plasmid** via PCR. In order to demonstrate our **engineering success**, we want to introduce you to one of the **most vital parts** of our expression system, the [**sAP secretion signal**](http://parts.igem.org/Part%3ABBa_K3781005).

The sAP signal tag is a **secretion signal peptide** derived from the secretory protein **secreted acid phosphatase 1 (sAP1)** which was originally purified from *Leishmania mexicana*, a human pathogenic *Leishmania* strain[[1].](#ref-1) It mediates the **exocytosis** of the protein after its biosynthesis in the endoplasmatic reticulum (ER). This is extremely useful for recombinant protein production, since target proteins can be **harvested** directly from **cell culture supernatant** without the need for cell lysis. Since **post-translational glycosylation** of proteins happens within ER and Golgi apparatus, along the **secretory pathway** [[2]](#ref-2), the sAP1 signal peptide enables the **production of glycosylated proteins** in *Leishmania*. This was a **vital aspect** to our project, because we aimed to demonstrate *Leishmania*’s **complex, human-like glycosylation capabilities** which make them such a promising **expression host** for **biopharmaceutical proteins**. During the realisation of this project, the **SARS-CoV-2 receptor binding domain (RBD)** functioned as our exemplary protein of interest. Not only would transgenic expression of the SARS-CoV-2 RBD prove our expression system’s **scientific and medical relevance**, it would also shine a light on the **post-translational modification capabilities** of *Leishmania*, since the receptor binding domain is a **heavily glycosylated** protein.

So now that you know how important protein secretion is to the objective of our project, it doesn’t come unexpected that most of the [**L1 constructs**](https://2021.igem.org/Team%3ATU_Kaiserslautern/Composite_Parts) we created with our [**MocloMania collection**](https://2021.igem.org/Team%3ATU_Kaiserslautern/Parts) contain the N-terminal sAP secretion signal. But how did we design it and construct it?

## DESIGN

Well, the sequence to this part was directly derived from the commercial **pLEXSY\_I-blecherry3** plasmid, distributed by **JenaBioscience**, which is the basisof our L1 expression vector [weird\_plex](http://parts.igem.org/Part%3ABBa_K3781105).
pLEXSY\_I-blecherry3 is a *Leishmania* expression vector that was specifically designed to allow for either **cytosolic** or **secretory protein expression**. The *Leishmania mexicana* derived **sAP secretion signal peptide** is thus **already included** in the plasmid's backbone. JenaBioscience constructed the cloning frame in a way that would allow for either **inclusion** or **exclusion** of the signal peptide from the **open reading frame**, depending on the **choice of restriction enzymes** used for target protein insertion[[3]](#ref-3).

During the **domestication** of pLEXSY\_I-blecherry3 for the **MoClo system**, we **introduced** a **LacZ-alpha** gene, but **eliminated** the **sAP secretion sequence**. Instead, the sequence was adapted and turned into a functional **L0 basic part**, the [sAP secretion signal](http://parts.igem.org/Part%3ABBa_K3781005) that we are looking at right now. This way, **switching** between cytosolic and secretory protein expression can be even more **easily coordinated**, simply by adding or leaving out parts in a **MoClo reaction**.

The very first step in our endeavour to create this part was to find its sequence within the pLEXSY\_I-blecherry3 backbone and double-check it with protein sequence data found in online resources such as *UniProt*, see *Figures 1* and *2*.

![](https://static.igem.org/mediawiki/parts/0/04/T--TU_Kaiserslautern--Engineering_UniProt.png)

Amino acid sequence of *Leishmania mexicana*'s **secreted acid phosphatase 1** with highlighted **secretion signal peptide** sequence | SOURCE | *UniProt*

![](https://static.igem.org/mediawiki/parts/e/e2/T--TU_Kaiserslautern--Engineering_pLEX_Sequence.png)

Genetic sequence and amino acid sequence of the sAP secretion signal peptide as found in the pLEXSY\_I-blecherry3 backbone

After confirming the exact position of the sequence within the pLEXSY\_I-blecherry3 backbone, we had to retrieve it from the plasmid. For this, we designed a **specified set of primers** called *sAP-forward* and *sAP-reverse* that would allow for **amplification** of the sAP sequence via PCR, while simultaneously introducing specified **four nucleotide overhangs** and **BbsI restriction sites** to the sequence. These additions are what makes the sequence suitable for application within our **Modular Cloning system**.
For the sAP secretion signal to be functional, it has to be attached to the target protein's very **N-terminal region**. Thus, we decided to adapt the part to **B2**, our **most upstream cloning position**. This meant that the respective overhangs that needed to be attached were *CCAT* to the **5' end** and *AATG* to the **3'** end of our sequence. For more information on primer design as well as overhang choice, feel free to check out our tutorial on [**how to**](https://2021.igem.org/Team%3ATU_Kaiserslautern/Contribution) generate a MoClo part.

**Table 1:** Table displaying compostion of the primers used for amplification and Moclo adaptation of the sAP signal peptide sequence

![](https://static.igem.org/mediawiki/parts/8/8c/T--TU_Kaiserslautern--Overhang_Table_Purple.png)

We tested our primer design by going through the entire **amplification** and **cloning process** ***in silico***. When we found everything to work out well, it was time to **order the primers**. We also set up an experimental plan for the upcoming weeks.

**EXPERIMENTAL PLAN**

**1** | PCR with ensuing gel electrophoresis and gel extraction

 **2** | MoClo ligation of extracted fragment into B2 plasmid backbone

 **3** | Transformation, plasmid prep and test digest to confirm successful insertion

 **4** | Assembly of resulting L0\_sAP\_B2 into L1 construct

 **5** | Transformation, plasmid prep and test digest to confirm successful assembly

 **6** | Transfection into *Leishmania tarentolae* cell culture

 **7** | Investigation of protein expression via immunostaining on Western blot

So upon **arrival of the primers**, we were ready to go!

## CONSTRUCTION

Right after arrival of the primers, we resuspended and diluted them to the desired concentration and then conducted a **standard PCR amplification** reaction using NEB's **Q5 Polymerase**. As a **template**, we used the unprocessed **pLEXSY\_I-blecherry3** plasmid. After completion of the thermocycling process, we loaded the resulting PCR sample onto a **2% agarose gel**, accompanied by the Thermo Fisher LowRange GeneRuler. We did this because thanks to our *in silico* amplification, we knew that we were expecting a small, **100 bp fragment**.

![](https://static.igem.org/mediawiki/parts/2/21/T--TU_Kaiserslautern--Engineering_sAP_PCRNEW.png)

Gel electrophoresis of PCR fragment
**1** | PCR *sAP-forward* + *sAP-reverse* on *pLEXSY\_I-blecherry3*
**L** | Thermofisher LowRange GeneRuler [bp]

We observed the predicted 100 bp fragment on the gel which meant that we could continue with our experimental plan. By **extracting** the **fragment** from the agarose gel with the help of a MACHEREY-NAGEL *Gel Extraction Kit* we recovered the PCR fragment, **free** from any **residual template DNA** or enzymes present in the PCR reaction. This allowed us to directly introduce the recovered fragment into a **MoClo ligation reaction** in order to insert it into [**pAGM1276**](http://parts.igem.org/Part%3ABBa_K3781100), the **B2 plasmid backbone** adapted from the **Weber MoClo plasmid collection** [[4].](#ref-4) Introduction into a plasmid backbone doesn't only preserve our fragment from **degradation**, it also helps us to evaluate whether the cloned PCR fragment was amplified accurately and really carries the **desired overhangs**.

As a means of demonstration, we have included a **standard MoClo Ligation** protocol that we used standardly when it came to generation of L0 basic parts. For a MoClo reaction to be efficient, it is important to add all genetic components, backbone as well as insert, in equal stoichiometric quantities. For many protocols this is **40 fmol**, but other values such as 20 fmol are published as well[[5]](#ref-5). Thanks to **photometry**, we were able to quantify the **concentrations** of our pAGM1276 as well as of our gel-excised sAP fragment. With the help of the **Bioline** [**online molar quantity calculator,**](https://www.bioline.com/media/calculator/01_07.html) this allowed us to predict the exact **sample volume** that needed to be added to the reaction mix in order to give **40 fmol sample molecules** each.

**L0 MoClo ligation** | 20 µL reaction volume

2 µL | **10x NEB CutSmart-Buffer**

2 µL | **ATP**

0,5 µL | **BbsI**

0,5 µL | **T4 DNA Ligase**

40 fmol | **sAP Fragment**

40 fmol | **pAGM1276**

fill up | **ddH2O**

After addition of all reactants (enzymes last), the reaction mix was **briefly vortexed**, **spun down** and then put into a **thermocycler** to undergo the standard **MoClo cycling process**. Every protocol does this a little differently, but the basic cycle program is always the same.

**L0 MoClo ligation** | Thermocycling program

**45x**
--------------------------------
**37°C** | 2 min | **16°C** | 5 min
--------------------------------
**1x**
--------------------------------
**37°C** | 5 min
**80°C** | 10 min
**16°C** | indefinitely
--------------------------------

After completion of the MoClo thermocycling run (usually performed **over night**), **10 µL** of the resulting sample were transformed into **competent TOP10 *E. coli***. This *E. coli* strain allows for rapid and efficient **amplification** of the hopefully ligated **L0 construct** and is also suitable for **blue-white screening**. Since our goal was to insert the **sAP sequence** into the **B2 backbone** pAGM1276, we will proceed to call the cloned construct **L0\_sAP\_B2**, according to our standardized **Moclo Mania nomenclature**.

After transformation of the sample and incubation of the transformed *E. coli* at 37°C over night on an **IPTG/XGal-agar plate**, white and blue colonies could be observed the next morning. Because the ***lacZ-alpha* gene fragment** in pAGM1276 is **excised** upon insertion of the sAP fragment, the resulting transformants don't have all the parts it takes to produce functional **beta-galactosidase**. This means that the **white colonies** are the ones carrying our **successfully cloned construct**. We inoculated several of them into liquid culture and let them grow on a shaker at 37°C over night. Both, the LB medium for the agar plates and that for the liquid culture had **spectinomycin** added, since this is the antibiotic that our **L0 plasmid backbones** carry a **resistance marker** against.

The next morning, the liquid cultures showed substantial growth and could be prepped with the help of a MACHEREY-NAGEL *DNA MiniPrep Kit*. This way, the replicated L0\_sAP\_B2 construct could be extracted from the *E. coli* cells and purified. We ended up with our purified, (hopefully) correctly assembled L0 construct in a microfuge tube on ice. Now, in order to **verify** that the DNA in our tube is really the construct we are looking for and not just some random *E. Coli* plasmid, we performed a **test digest**. Again, going through this process *in silico* was very helpful to us, because it allowed us to easily plan the most feasible digest, rendering the **most distinct band pattern** and only requiring **easily applicable enzymes** with high activity in the same buffer. We finally settled on a digest using **BstEII** as well as **XhoI**. Doing a **double digest** like this was particularly handy, because XhoI cuts **within the actual sAP sequence**. This means that the **successfully cloned construct** would result in **two bands**, whereas the **empty pAGM1276 backbone** could only be **linearized**.

**Test digest** | 20 µL reaction volume

2 µL | **10x NEB CutSmart-Buffer**

0,75 µL | **BstEII**

0,75 µL | **XhoI**

500 ng | **DNA sample**

fill up | **ddH2O**

Incubate at 37°C for 1 h.

![](https://static.igem.org/mediawiki/parts/4/46/T--TU_Kaiserslautern--Engineering_Gelbild_L0_sAP_B2NEW.png)

Test digest of L0-sAP\_B2 using *BstEII* and *XhoI*
**1** | [pAGM1276](http://parts.igem.org/Part%3ABBa_K3781102) | 2842 bp
**2** | **L0\_sAP\_B2** | 1705 + 615 bp
**L** | Thermo Fischer GeneRuler Plus Ladder [bp]

As can be seen in *Figure 4*, the empty **pAGM1276** shows **one band**, whereas the digested **L0\_sAP\_B2** displays the exact **two band pattern** that we anticipated from our *in silico* digest. This is already a good indicator that our L0 part was **cloned successfully**! But what if point mutations were introduced during PCR, too small to be seen with gel electrophoresis? In order to dispel any doubts and verify the **correct insertion** and **sequence** of our **sAP secretion peptide** once and for all, we sent the plasmid off to commercial **Sanger sequencing** at MICROSYNTH, a Swiss-based sequencing company with a branch in Germany.

For this, we relied on a set of **sequencing primers** that have been designed by our **supervising working group** in order to facilitate sequencing of L0 basic parts. Due to the **high similarity** between the indivdual **L0 plasmid backbones** (they only really differ at the **insertion site**), these primers called *L0-Seq-forward* and *L0-Seq-reverse* can be used for sequencing of any and all L0 parts, **independently** of their **cloning position**.

*L0-Seq-forward* | aatacgcaaaccgcctctcc
*L0-Seq-reverse* | aataggcgtatcacgaggc

![](https://static.igem.org/mediawiki/parts/a/a6/T--TU_Kaiserslautern--Engineering_Sequencing_Result.png)

Aligned sequences of *in silico* cloned L0\_sAP\_B2 and sample containing L0\_sAP\_B2 + *L0-Seq-forward*. The black arrows mark the beginning and the end of our B2 sAP secretion signal.

Only three days after sending our sample off to sequencing, we got the result! It's a bit like opening a bag of wonders every time you click on those sequencing files. You just never know wether things are going to allign perfectly or wether nothing seems to match at all! Luckily, our L0\_sAP\_B2 looked very good, as you can see in *Figure 5*. The sequence of the processed *L0-Seq-forward* initiated fragment matches that of the *in silico* cloned L0\_sAP\_B2 perfectly, except for usual sequencing uncertainties in the very terminal regions of the fragment. This was great news!! Now we could continue our experimental journey by assembling our L0 secretion signal into a variety of L1 constructs. Thanks to the practicability of Modular Cloning, this wasn't a very difficult endeavour.

Now we have already elaborated on the **great importance** of the sAP secretion signal to the prospect of our project, so it doesn't come as a surprise that as soon as we had the finished L0\_sAP\_B2 part, we immediately started **implementing** it into **all of our L1 constructs**. In order to simplify this demonstration, let's just look at the MoClo ligation reaction performed for generating [**L1\_sAP\_RBD\_Strep8His**](http://parts.igem.org/Part%3ABBa_K3781213). This L1 composite part contains the **sAP secretion signal**, a coding sequence for the [**SARS-CoV-2 receptor binding domain**](http://parts.igem.org/Part%3ABBa_K3781001) and a C-terminal [**Strep8His**](http://parts.igem.org/Part%3ABBa_K3781017) purification tag.

You will see that the reaction mix is basically identical to that of the **L0 ligation**, except now we have to remember the **two-enzyme system** of MoClo and employ **BsaI** as our executing type IIS restriction enzyme. Also, instead of a L0 plasmid backbone we now want to include our L1 expression vector, [**weird\_plex**](http://parts.igem.org/Part%3ABBa_K3781105), that will allow us to transfect and express our construct within *Leishmania tarentolae*. **Thermocycling conditions** for this ligation are virtually identical to the ones for a L0 ligation. The only exception is that, after cycling, a **5 min incubation** is performed at **50°C** rather than **37°C**, because BsaI has a slighlty different activity pattern than BbsI.

**L1 MoClo ligation** | 20 µL reaction volume

2 µL | **10x NEB CutSmart-Buffer**

2 µL | **ATP**

0,5 µL | **BsaI**

0,5 µL | **T4 DNA Ligase**

40 fmol | **L0\_sAP\_B2**

40 fmol | **L0\_RBD\_B3\_B4**

40 fmol | **L0\_Strep8His\_B5**

40 fmol | **weird\_plex**

fill up | **ddH2O**

Again, after completion of the MoClo thermocycling run, **10 µL** of the resulting sample were transformed into **competent TOP10 *E. coli***. And yet again, after over-night incubation on **IPTG/Xgal agar plates**, we inoculated the **white colonies** into liquid culture. This time, though, both the LB medium for the agar plates as well asand that for the liquid culture had **ampicillin** added, because **weird\_plex** carries its own ***E. coli* expression cassette** that includes an **ampicillin resistance marker**.

The next morning, the liquid cultures were again prepped with the help of the MACHEREY-NAGEL *DNA MiniPrep Kit* and the (hopefully) assembled L1\_sAP\_RBD\_Strep8His construct was extracted from the *E. coli* cells. To **verify** that our construct was correctly assembled, we performed another **test digest**. *In silico* cloning and digestion helped us to choose **PstI**, because it gives a very nice and distinct band pattern.

![](https://static.igem.org/mediawiki/parts/8/82/T--TU_Kaiserslautern--Engineering_Gelbild_L1_sAP_RBD_Strep8His.png)

**Test digest of L1\_sAP\_RBD\_Strep8His** using *PstI*
**1** | **L1\_sAP\_RBD\_Strep8His** | 3894 + 1323 + 991 + 991 + 757 bp
**L** | Thermo Fischer GeneRuler Plus Ladder [bp]

As you can see in **Figure 6**, our test digest on the L1 construct resulted in the expected bands. This means that the plasmid in our tube is of the predicted size, which is a very good sign, since this probably means that all L0 parts have been assembled into the L1 backbone successfully. To rule out any assembly errors and to **verify** the identity of our prepped L1 construct with 100% certainty, we again sent the plasmid off to **Sanger sequencing** at [Microsynth.](https://www.microsynth.com/home-de.html "Microsynth")

![](https://static.igem.org/mediawiki/parts/3/3e/T--TU_Kaiserslautern--Engineering_Sequencing_L1%21%21.png)

Sequence alignment for sequenced L1\_sAP\_RBD\_Strep8His composite part compared to *in silico* part

Yayy! After three days, the sequencing results are back and the alignment looks super sharp! Except from a few bases that were probably missed due to clustering of identical bases (Sanger sequencing always struggles with **repetitive single nucleotide sequences**), everything looks super promising. This means that we not only successfully constructed our basic **L0\_sAP\_B2** part, but we have also managed to assemble it into a **complete L1 construct**. Now, as mentioned before, we of course didn't only do this for L1\_sAP\_RBD\_Strep8His, but for many other constructs that are all ready for the next experimental round. Transfection into *Leishmania*!

## EXPRESSION

Now that we have mastered all the cloning work, it is time to move on to the second big step in our project - expressing the genetic constructs in ***Leishmania tarentolae***. To do this, we of course have to somehow get our construct **into** *Leishmania* cell first. How do we do this? By **transfecting** the cells via **electroporation**. This procedure as well as all other labwork involving the cultivation of *Leishmania* is based on the protocols provided by our second **supervising working group** as well as the protocols distributed by **JenaBioscience** in their commercial [**LEXSinduce expression kit**](https://www.jenabioscience.com/lexsy-expression/lexsy-configurations/inducible-genome-integrated/ege-1410blecherry-inducible-lexsy-expression-kit). If you are interested in finding out more about the specifics of working with *Leishmania*, please feel free to check out our guide on [**how to**](https://2021.igem.org/Team%3ATU_Kaiserslautern/Contribution) handle *Leishmania*!

After transfecting the parasites with our L1\_sAP\_RBD\_Strep8His, the *Leishmania* are brought out onto agar plates. Here, addition of the antibiotic **bleomycin** ensures that all growing colonies must have taken up our plasmid, since the L1 expression vector **weird\_plex** is equipped with a **bleomycin resistance gene**. It takes anything from **5 to 9 days** for colonies of sufficient size to form on the plate. These can then be **inoculated** into **liquid medium** where the parasites can be grown and **re-diluted** into ever larger volumes. Once the cell culture has reached the desired density, **protein expression** can be **induced** by addition of **tetracycline** to the culture medium. For more in-depth information on the specific features of the weird\_plex expression vector and its inducible expression, feel free to head over to the [**weird\_plex**](http://parts.igem.org/Part%3ABBa_K3781105) part page.

Tetracycline induction is held stable for around **2-3 days**, until the cell culture is finally **harvested**. By **spinning down** the culture in a centrifuge for over an hour, the cells are separated from the **culture medium** and form a **pellet**. Depending on the **localisation** of the expressed protein, this pellet can be lysed and the resulting **lysate** either **purified** (cytosolic expression) or **cooked up** with **SDS sample buffer** for analysis (secreted expression). In our case most of the L1 constructs, including **L1\_sAP\_RBD\_Strep8His**, mediate **secretion** of the **recombinantly expressed protein** into the culture medium. Thus, after centrifugation, the cell culture **supernatant** is **carefully collected** in order to immediately undergo downstream **purification procedures**. Only a small sample is saved for analysis purposes. The sample is treated with **trichloroaceticic acid** in order to **precipitate** all protein and the **resulting pellet** cooked up with **SDS sample buffer**. Along with the cell lysate sample as well as **control samples** of **untransfected *Leishmania* cell culture**, it is loaded onto an SDS-gel for **SDS-PAGE**. After **western blotting**, the samples can be **immunostained** with the help of different antibodies in order to visualize the target protein. In the case of **L1\_sAP\_RBD\_Strep8His**, we chose **antibodies against RBD** to stain against our fusion protein.

![](https://static.igem.org/mediawiki/parts/e/e8/T--TU_Kaiserslautern--Engineering_Blot_L1-sAP_RBD_Strep8His.png)

**Immunoblot** | of Leishmania cell cultures transfected with different L1 constructs
1. AB | **ms anti-RBD**
2. AB | **rb anti-ms HRP**
**1** | **L1\_sAP\_RBD\_Strep8His** | 27.7 kDa
**2** | [L1\_sAP\_Myc\_RBD\_Strep8His](http://parts.igem.org/Part%3ABBa_K3781204) | 30.2 kDA
**3** | [L1\_sAP\_RBD\_GST](http://parts.igem.org/Part%3ABBa_K3781214) | 51.9 kDa
**n.c.** | negative control | untransfected Leishmania culture
**P** cell pellet  |  **S** cell culture supernatant  |  **+** culture induced with tetracycline  |
**L** | Thermo Fischer PageRuler Protein Ladder [kDa]

![](https://static.igem.org/mediawiki/parts/3/3d/T--TU_Kaiserslautern--Engineering_Blot_L1_sAP_VS_3xHA.png)

**Immunoblot** | of Leishmania cell cultures transfected with different L1 constructs
1. AB | **ms anti-RBD**
2. AB | **rb anti-ms HRP**
**1** | [L1\_sAP\_RBD\_mVenus](http://parts.igem.org/Part%3ABBa_K3781210) | 52.1 kDa
**2** | [L1\_sAP\_RBD\_mVenus\_GST](http://parts.igem.org/Part%3ABBa_K3781212) | 78.8 kDA
**3** | [L1\_3xHA\_RBD\_mVenus](http://parts.igem.org/Part%3ABBa_K3781201) | 55.9 kDa
**4** | [L1\_3xHA\_RBD\_HA8His](http://parts.igem.org/Part%3ABBa_K3781206) | 28 kDa
**p.c.** | positive control | RBD-GFP fusion protein | 54 kDa
**n.c.** | negative control | Leishmania culture transfected with empty weird\_plex
**P** cell pellet  |  **S** cell culture supernatant  |  **+** culture induced with tetracycline  |
**L** | Thermo Fischer PageRuler Protein Ladder [kDa]

After loading several different cell cultures transfected with our **secreted fusion proteins** onto a blot and staining against the **SARS-CoV-2 receptor binding domain**, see *Figure 8*, we detected a **significant expression** of our recombinant protein in **all three transfected cell lines**, *1-3*! As expected, the **negative control** *n.c.*, a *Leishmania* culture that wasn't transfected with any L1 expression vector, showed no bands. Furthermore, protein expression could only be detected in cultures that were induced with tetracycline, *P+/S+*, advocating the efficiency of the **tet-controlled transcriptional activation**. Looking back at our sAP secretion tag, we can observe that bands of the expected size appear in both the cell lysate *P* as well as the culture supernatant *S*, but **signal intensity** is much **higher** in the **supernatant** for all three cultures. The faint bands in the lysate samples indicate that a small part of our fusion protein might still be on the way out of the cells. Yet, the **vast majority** of protein is **successfully secreted** into the culture medium! The same holds true for cultures *1* and *2* in *Figure 9* where protein expression was only detected in the cell culture supernatant. This proves the **functionality** of our **sAP secretion signal** and highlights its importance in producing complexly glycosylated protein that need to **pass the secretory pathway**.

## PURIFICATION

After expression in *Leishmania*, we of course also wanted to **harvest** the fruit of our work, our **secreted recombinant protein**. The best way to do this is to incorporate a **purification tag** into the fusion protein, ideally allowing us to purify it directly from crude cell culture supernatant with the help of **gravity flow/batch purification** procedures. These procedures certainly hold the most room for **improvement** when it comes to the **ongoing implementation** of our project, since many purification attempts have remained **unsuccessful**. We are still working hard on figuring out what factors can be altered in order to increase purification yield. Since Strep-purification has not given good results yet, we cannot continue our demonstration by following **L1\_sAP\_RBD\_Strep8His** all the way down the line. Instead we'll take a look at a GST-tagged fusion protein, [**L1\_sAP\_RBD\_GST**](http://parts.igem.org/Part%3ABBa_K3781214), that shows the most promising results and gives further evidence to the **quality** of our **sAP secretion signal**!

![](https://static.igem.org/mediawiki/2021/7/74/T--TU_Kaiserslautern--Engineering_success.jpg)

**Immunoblot | of Leishmania cell culture after GST-purification**
culture was transfected with **L1\_sAP\_RBD\_GST** and protein expression **induced** with tetracycline
blot was co-stained against both **RBD** and **GST** a) Culture supernatant b)Lysate after cell debris

There are two things that we can conclude from the blots in *Figure 10*. First, there is an **issue** with our **GST-tag** being **cleaved off prematurely**, rendering a lot of singular GST and decreasing the purification yield of our **RBD\_GST fusion protein**. Second, there are **differences** between the samples purified from the **cell lysate** (a) and the ones purified from the **culture medium supernatant** (b). We can see that the **intensity** of the immunostain is much **higher** in the **supernatant purification**, suggesting that **recombinant protein quantities** in the culture medium exceeded those of the cell lysate by far. Thus, our **sAP secretion tag** effectively mediates translocation of the target protein into the cell's exterior!

Finally, we could not only show that the **sAP secretion peptide** has been successfully adapted as a **MoClo basic part**, which can be assembled into **cohesive L1 constructs**, we were also able to show that it can be transgenically expressed in *Leishmania tarentolae* and mediates **exocytosis** of the recombinant target protein into the surrounding **cell culture medium**.
Now of course, most of our cloning, expression and purification efforts didn't show complete success upon first trial, so keep in mind that for many of our parts, including L0\_sAP\_B2, the generation process was a lot more tedious than the idealized description given above. By tweaking parameters such as **thermocycling settings** during PCR or **DNA concentrations** during ligation, most of these obstacles were eventually overcome and led us to build the extensive [**part collection**](https://2021.igem.org/Team%3ATU_Kaiserslautern/Parts) that we are proud to present to you today! Feel free to check out our [**lab journal**](https://2021.igem.org/Team%3ATU_Kaiserslautern/experiments) to catch a first-hand view on our experimental approaches. And as always, reach out to us if you have any further questions!

### Sources

1. [**Ilg T**, Stierhof YD, Etges R, Adrian M, Harbecke D, Overath P. Secreted acid phosphatase of Leishmania mexicana: a filamentous phosphoglycoprotein polymer. Proc Natl Acad Sci U S A. 1991;88(19):8774-8778. doi:10.1073/pnas.88.19.8774](http://europepmc.org/article/PMC/52592)
2. [**Schjoldager, K.T.**, Narimatsu, Y., Joshi, H.J. et al. Global view of human protein glycosylation pathways and functions. Nat Rev Mol Cell Biol 21, 729–749 (2020). https://doi.org/10.1038/s41580-020-00294-x](https://pubmed.ncbi.nlm.nih.gov/33087899/)
3. [https://www.jenabioscience.com/files/jenabioscience/datasheet\_extern/EGE-1410.pdf, pages 9, 18, last visited 10/17/21, 11:00 ECT](https://www.jenabioscience.com/files/jenabioscience/datasheet_extern/EGE-1410.pdf)
4. [**Weber E**, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE 6(2): e16765. https://doi.org/10.1371/journal.pone.0016765](https://www.tandfonline.com/doi/pdf/10.4161/bbug.3.1.18223)
5. [**Marillonnet, S.**, & Grützner, R. (2020). Synthetic DNA assembly using golden gate cloning and the hierarchical modular cloning pipeline. Current Protocols in Molecular Biology, 130, e115. doi: 10.1002/cpmb.115](https://www.semanticscholar.org/paper/Synthetic-DNA-Assembly-Using-Golden-Gate-Cloning-Marillonnet-Gr%C3%BCtzner/305727126639960cda906a50876728b195da6689)

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**2021**

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# Team:TU Kaiserslautern/Experiments

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[MOCLO MANIA](https://2021.igem.org/Team%3ATU_Kaiserslautern)

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[School Presentation](#head-1)

# EXPERIMENTS

## Basic Protocols

Leishmania
![](https://static.igem.org/mediawiki/2021/9/95/T--TU_Kaiserslautern--buch.png)

Read

[* BHI agar medium](https://static.igem.org/mediawiki/2021/e/e2/T--TU_Kaiserslautern--BHI-agar.pdf)
[* BHI freezing medium](https://static.igem.org/mediawiki/2021/7/79/T--TU_Kaiserslautern--BHI_freeze.pdf)
[* BHI medium original cells](https://static.igem.org/mediawiki/2021/e/e6/T--TU_Kaiserslautern--BHI_ori.pdf)
[* BHI medium after transfection](https://static.igem.org/mediawiki/2021/4/4b/T--TU_Kaiserslautern--BHI_transf.pdf)
[* Buffers](https://static.igem.org/mediawiki/2021/3/37/T--TU_Kaiserslautern--Buffers.pdf)
[* Culture preparation for purification](https://static.igem.org/mediawiki/2021/3/31/T--TU_Kaiserslautern--Cell_prep_puri.pdf)
[* Dilution of cell cultures](https://static.igem.org/mediawiki/2021/d/d1/T--TU_Kaiserslautern--Dilution.pdf)
[* Freezing cell cultures](https://static.igem.org/mediawiki/2021/2/23/T--TU_Kaiserslautern--Freezing.pdf)
[* Immunoblot](https://static.igem.org/mediawiki/2021/8/85/T--TU_Kaiserslautern--Immunoblot.pdf)
[* Tetracycline Induction](https://static.igem.org/mediawiki/2021/a/a9/T--TU_Kaiserslautern--Induction.pdf)
[* mCherry spectrum](https://static.igem.org/mediawiki/2021/0/06/T--TU_Kaiserslautern--mCherry_spectrum.pdf)
[* Picking clones](https://static.igem.org/mediawiki/2021/5/54/T--TU_Kaiserslautern--Picking.pdf)
[* Plating out cell cultures](https://static.igem.org/mediawiki/2021/1/1b/T--TU_Kaiserslautern--Plating.pdf)
[* Stripping membranes](https://static.igem.org/mediawiki/2021/1/1b/T--TU_Kaiserslautern--Plating.pdf)
[* Transfection](https://static.igem.org/mediawiki/2021/f/f3/T--TU_Kaiserslautern--Transfection.pdf)
[* Transfection Buffer](https://static.igem.org/mediawiki/2021/7/77/T--TU_Kaiserslautern--Transfection_Buffer.pdf)

MoClo
![](https://static.igem.org/mediawiki/2021/9/95/T--TU_Kaiserslautern--buch.png)

Read

[* 1% HD Green Agarose gel](https://static.igem.org/mediawiki/2021/d/d1/T--TU_Kaiserslautern--1%25_gel.pdf)
[* 2% HD Green Agarose gel](https://static.igem.org/mediawiki/2021/7/7b/T--TU_Kaiserslautern--2%25_gel.pdf)
[* 50x TAE](https://static.igem.org/mediawiki/2021/f/fe/T--TU_Kaiserslautern--50x_TAE.pdf)
[* Competent Making](https://static.igem.org/mediawiki/2021/8/80/T--TU_Kaiserslautern--Competent_Making.pdf)
[* Diagnostic Digest](https://static.igem.org/mediawiki/2021/8/80/T--TU_Kaiserslautern--Competent_Making.pdf)
[* DMSO Stock](https://static.igem.org/mediawiki/2021/8/87/T--TU_Kaiserslautern--DMSO.pdf)
[* DNA precipitation](https://static.igem.org/mediawiki/2021/a/a2/T--TU_Kaiserslautern--DNA_precip.pdf)
[* Gel Extraction](https://static.igem.org/mediawiki/2021/3/35/T--TU_Kaiserslautern--GelEx.pdf)
[* HD Green Poststain](https://static.igem.org/mediawiki/2021/7/7e/T--TU_Kaiserslautern--Poststain.pdf)
[* Inoculation](https://static.igem.org/mediawiki/2021/3/38/T--TU_Kaiserslautern--Inoculation.pdf)
[* LB agar plates](https://static.igem.org/mediawiki/2021/9/9c/T--TU_Kaiserslautern--LB_agar.pdf)
[* Modular Cloning](https://static.igem.org/mediawiki/2021/d/d0/T--TU_Kaiserslautern--MoClo_Prot.pdf)
[* Plasmid Miniprep](https://static.igem.org/mediawiki/2021/f/f5/T--TU_Kaiserslautern--Miniprep.pdf)
[* PCR using Q5 DNA Polymerase](https://static.igem.org/mediawiki/2021/3/3c/T--TU_Kaiserslautern--Q5_PCR.pdf)
[* Ligation using T4 DNA Ligase](https://static.igem.org/mediawiki/2021/f/ff/T--TU_Kaiserslautern--T4_Ligation.pdf)
[* TOP10 E. coli Transformation](https://static.igem.org/mediawiki/2021/f/ff/T--TU_Kaiserslautern--T4_Ligation.pdf)

Protein
![](https://static.igem.org/mediawiki/2021/9/95/T--TU_Kaiserslautern--buch.png)

Read

[* Antibody staining](https://static.igem.org/mediawiki/2021/1/1c/T--TU_Kaiserslautern--Antibody.pdf)
[* Ammonium sulfate precipitation](https://static.igem.org/mediawiki/2021/b/bf/T--TU_Kaiserslautern--ASP.pdf)
[* GST Purification](https://static.igem.org/mediawiki/2021/c/c1/T--TU_Kaiserslautern--GST.pdf)
[* His Purification](https://static.igem.org/mediawiki/2021/b/b8/T--TU_Kaiserslautern--His.pdf)
[* Chemiluminescence Detection using HRP](https://static.igem.org/mediawiki/2021/7/7f/T--TU_Kaiserslautern--HRP.pdf)
[* SDS gels](https://static.igem.org/mediawiki/2021/c/c2/T--TU_Kaiserslautern--SDS_gels.pdf)
[* Strep Purification](https://static.igem.org/mediawiki/2021/c/c3/T--TU_Kaiserslautern--Strep.pdf)
[* TCA precipitation](https://static.igem.org/mediawiki/2021/8/83/T--TU_Kaiserslautern--TCA.pdf)
[* Western Blot 1](https://static.igem.org/mediawiki/2021/e/e3/T--TU_Kaiserslautern--WB_MiBi.pdf)
[* Western Blot 2](https://static.igem.org/mediawiki/2021/f/fa/T--TU_Kaiserslautern--WB_Biotech.pdf)
[* Preparing human cells for Western Blot](https://static.igem.org/mediawiki/2021/a/a1/T--TU_Kaiserslautern--human_cells.pdf)

## Labjournal

Leishmania
![](https://static.igem.org/mediawiki/2021/9/95/T--TU_Kaiserslautern--buch.png)
[Read](https://static.igem.org/mediawiki/2021/2/26/T--TU_Kaiserslautern--Leishmania_LAB.pdf)

MoClo
![](https://static.igem.org/mediawiki/2021/9/95/T--TU_Kaiserslautern--buch.png)
[Read](https://static.igem.org/mediawiki/2021/e/ea/T--TU_Kaiserslautern--MoClo_LAB.pdf)

Protein
![](https://static.igem.org/mediawiki/2021/9/95/T--TU_Kaiserslautern--buch.png)
[Read](https://static.igem.org/mediawiki/2021/4/4c/T--TU_Kaiserslautern--Protein_purification_LAB.pdf)

Activity Assay
![](https://static.igem.org/mediawiki/2021/9/95/T--TU_Kaiserslautern--buch.png)
[Read](https://static.igem.org/mediawiki/2021/5/5b/T--TU_Kaiserslautern--Activity_assay_LAB.pdf)

[![](https://static.igem.org/mediawiki/2021/d/dd/T--TU_Kaiserslautern--LogoImplen.jpg)](https://2021.igem.org/Team%3ATU_Kaiserslautern/Sponsors#implen)

[![](https://static.igem.org/mediawiki/2021/e/ea/T--TU_Kaiserslautern--jenabioscience.png)](https://2021.igem.org/Team%3ATU_Kaiserslautern/Sponsors#jena)

[![](https://static.igem.org/mediawiki/2021/5/56/T--TU_Kaiserslautern--LogoSnapgene.png)](https://2021.igem.org/Team%3ATU_Kaiserslautern/Sponsors#snapgene)

[![](https://static.igem.org/mediawiki/2021/5/55/T--TU_Kaiserslautern--LogoLabforward.png)](https://2021.igem.org/Team%3ATU_Kaiserslautern/Sponsors#labforward)

[![](https://static.igem.org/mediawiki/2021/d/d6/T--TU_Kaiserslautern--LogoHeidolph_Instruments.jpg)](https://2021.igem.org/Team%3ATU_Kaiserslautern/Sponsors#heidolph)

[![](https://static.igem.org/mediawiki/2021/c/c9/T--TU_Kaiserslautern--LogoSWK.jpg)](https://2021.igem.org/Team%3ATU_Kaiserslautern/Sponsors#stadtwerke)

iGEM Kaiserslautern

**2021**

*made by [jules](https://stonedstreet.de)*

[Website](https://www.uni-kl.de/hochschulgruppen/iGEM/en/hello/)
Facebook
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igem@bio.uni-kl.de

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:TU Kaiserslautern/Results

[![](https://static.igem.org/mediawiki/2021/c/c4/T--TU_Kaiserslautern--logo.png)](https://2021.igem.org/Team%3ATU_Kaiserslautern)

[MOCLO MANIA](https://2021.igem.org/Team%3ATU_Kaiserslautern)

* [Home](https://2021.igem.org/Team%3ATU_Kaiserslautern)
* Team
  + [Team](https://2021.igem.org/Team%3ATU_Kaiserslautern/Team)
  + [Attributions](https://2021.igem.org/Team%3ATU_Kaiserslautern/Attributions)
* Project
  + [Description](https://2021.igem.org/Team%3ATU_Kaiserslautern/Description)
  + [Implementation](https://2021.igem.org/Team%3ATU_Kaiserslautern/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3ATU_Kaiserslautern/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3ATU_Kaiserslautern/Results)
  + [Notebook](https://2021.igem.org/Team%3ATU_Kaiserslautern/Notebook)
  + [Model](https://2021.igem.org/Team%3ATU_Kaiserslautern/Model)
* Laboratory
  + [Engineering](https://2021.igem.org/Team%3ATU_Kaiserslautern/Engineering)
  + [Contribution](https://2021.igem.org/Team%3ATU_Kaiserslautern/Contribution)
  + [Experiments](https://2021.igem.org/Team%3ATU_Kaiserslautern/Experiments)
  + [Safety](https://2021.igem.org/Team%3ATU_Kaiserslautern/Safety)
* Part Collection
  + [MoClo Mania Collection](https://2021.igem.org/Team%3ATU_Kaiserslautern/Parts)
  + [Composite Parts](https://2021.igem.org/Team%3ATU_Kaiserslautern/Composite_Parts)
* How To
  + [MoClo](https://2021.igem.org/Team%3ATU_Kaiserslautern/HowToMoClo)
  + [Leishmania](https://2021.igem.org/Team%3ATU_Kaiserslautern/HowToLeishmania)
* Human Practices
  + [Human Practices](https://2021.igem.org/Team%3ATU_Kaiserslautern/Human_Practices)
  + [Education and Communication](https://2021.igem.org/Team%3ATU_Kaiserslautern/Education)
  + [Collaborations](https://2021.igem.org/Team%3ATU_Kaiserslautern/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ATU_Kaiserslautern/Partnership)
* [Sponsors](https://2021.igem.org/Team%3ATU_Kaiserslautern/Sponsors)
* [Awards](https://2021.igem.org/Team%3ATU_Kaiserslautern/Awards)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3781)

[Introduction](#head-1)
[Domestication of Vector](#head-2)
[Cloning of parts](#head-3)
[Transfection](#head-4)
[Protein detection](#head-5)
[Protein purification](#head-6)
[Activity assay](#head-7)
[Outlook](#head-8)

# Results

## Introduction - Design of the constructs for the MoClo library

The goal of our project was to use the Modular Cloning system (MoClo)[[1]](#ref-1) to assemble gene constructs and introduce them into *Leishmania tarentolae* for protein production. For this, we created a library of 40 MoClo constructs for iGEM, consisting of one level-1 expression vector, 19 level-0 parts, and 20 level-1 constructs (the most important ones are shown in **Figure 1**). Constructs were propagated in TOP10 *Escherichia coli*, as this strain allows efficient cloning and high plasmid replication. The final level-1 constructs were transfected into *Leishmania tarentolae*. For this purpose, the *Leishmania* host strain T7-TR from Jena Bioscience was used, which belongs to the family of inducible LEXSY expression vectors. The pLEXSY\_I-blecherry3 expression vector was domesticated for our *L. tarentolae* MoClo System. The finished expression vector was then used together with the respective level-0 parts to assemble level-1 constructs. These were cut with the restriction enzyme SwaI for transfection and integration into the *Leishmania* genome.

![](https://static.igem.org/mediawiki/2021/c/cc/T--TU_Kaiserslautern--l1parts.jpg)

**Overview of the created Level 1 MoClo constructs**

14 of the level-1 constructs were eventually transfected into *Leishmania tarentolae*. The constructs start either with a secretion signal (sAP1) or a variable tag (e.g.: 3xHA). They are further composed of coding sequences for the proteins of interest (RBD, mCerulean, mVenus), have various purification tags (Strep8His, TEV-GST, Strep-Prec, GST-Prec, 8HisPrec and HA8His), tags for detection with commercial antibodies (Strep8His, TEV-GST, Strep-Prec, GST-Prec, 8His-Prec, Myc-Prec, 3xFlag-Prec and HA8His), and fluorescent proteins (mCerulean, mVenus). The modular cloning system provides variability in the assembly of constructs and allows for easy expansion of the part library.

As a protein of interest, we chose the SARS-CoV-2 receptor binding domain (RBD). Because *Leishmania tarentolae* shows human-like glycosylation patterns[[2]](#ref-2), constructs with a sAP1 (secreted acid phosphatase 1) secretion signal were assembled such that the produced RBD is transported out of the cell into the medium. Secretion is necessary for glycosylation. Constructs without a secretion signal were built to demonstrate that the protein can also be produced in the cytosol of *Leishmania* where it is not glycosylated. The different tags selected for detection and purification were used to make the MoClo library as diverse as possible.

## Domestication of vector

The first step for creating a functional expression system was to find a suitable expression vector that would allow for insertion of the desired gene constructs and efficient transfection into *Leishmania tarentolae*. For this, we relied on the “LEXSinduce3 Expression Kit”, distributed by Jena Bioscience GmbH, which is specifically designed to be used for heterologous protein expression in *Leishmania*. The vector contained in the kit is named pLEXSY\_I-blecherry3 and carries a bleomycin resistance marker for selection as well as mCherry fused to the marker for the visualization of transfected cells [[3].](#ref-3) In order to utilize pLEXSY\_I-blecherry3 as a plasmid backbone for Modular Cloning, any pre-existing BsaI recognition sites had to be removed from the plasmid to avoid fragmentation of the backbone during cloning. This was done by introducing single point mutations into the sites via PCR. New BsaI recognition sites at the 5’ and 3’ ends of the resulting PCR fragments should allow for their easy reassembly into a fully domesticated plasmid backbone via a standard MoClo reaction.

![]( https://static.igem.org/mediawiki/2021/0/05/T--TU_Kaiserslautern--Domestication_Results1.jpg)

**Concept of domestication of pLEXSY\_I-blecherry3 (A) to weird\_plex (B)**. Highlighted are the BsaI recognition sites to be removed from the plasmid (A, red) as well as the two BsaI recognition sites to be introduced into the plasmid, serving as entry site for MoClo assemblies (B, green). Shown are all important features of the expression vector, as well as the primers used for domestication.

Figure 2 shows the plasmid map of our expression vector weird\_plex. It contains all the necessary features for serving its purpose in our MoClo system, with an ampicillin resistance gene for transformation and propagation of the plasmid in *E. coli*, a *lacZ-alpha* gene cassette for blue-white screening in *E. coli*, a bleomycin resistance marker for selection of transfected *L. tarentolae* cells as well as a mCherry gene enabling monitoring of expression after induction.

The domestication of pLEXSY\_I-blecherry3 proved to be far more difficult than initially anticipated. In numerous attempts of PCR amplification, one of the fragments repeatedly turned out to be flawed, hindering successful creation of the final vector. After several unsuccessful attempts at amplifying the desired three PCR fragments, we decided to divide the problematic fragment into several smaller parts. Further attempts with new primers revealed a more detailed view onto the problem. After identifying a problematic 400-bp sequence spanned by primers plex-400-rev and plex-2-for, we decided on trying to amplify the sequence again which led to promising results, as shown in Figure 3.

![](https://static.igem.org/mediawiki/2021/e/e8/T--TU_Kaiserslautern--Domestication_results_2.jpg)

**Agarose gel electrophoresis of resulting fragments from domestication-PCR.**
Shown are the different band patterns after PCR with the respective primer combinations. Ladder: Thermo Fisher GeneRuler DNA Ladder Mix; Expected fragment sizes:
plex\_3-forward + plex\_1-reverse: 1886 bp
plex\_1-forward + plex\_2-reverse: 1718 bp
plex\_2-forward + plex\_400-reverse: 475 bp
plex\_2-forward-verlängert + plex\_400-reverse: 480 bp
plex\_400-forward + plex\_J-reverse: 1877 bp
plex\_400-forward + plex\_N-reverse: 1623 bp
plex\_J-forward + plex\_3-reverse: 2221 bp
plex\_N-forward + plex\_3-reverse: 2638 bp

Analysis of the underlying DNA sequences revealed a highly repetitive region (Fig 2A; labelled FU) within the fragment in question, suggesting possible difficulties of Q5 DNA polymerase with replicating them. Sequencing showed that there were about 150 bp missing in that fragment.

![]( https://static.igem.org/mediawiki/2021/8/86/T--TU_Kaiserslautern--Domestication_Results_Sequencingalignment.jpg)

**Results of Sanger sequencing of our weird\_plex expression vector.** (A) vector with 150 bp missing, (B) fully intact vector without missing base pairs.

Figure 4 shows the sequencing results that revealed the missing 150 bp containing a highly repetitive region between bp 1990 and bp 2030.

We tried to amplify the sequence from another template and for this used the pLEXSY\_IE-blecherry4 expression vector from Jena Bioscience GmbH. The 400 bp fragment, which had to be amplified out of the plasmid, was homologous to the sequence on our destination vector.

![]( https://static.igem.org/mediawiki/2021/0/0c/T--TU_Kaiserslautern--Results_domestication_4.jpg)

**Agarose gel electrophoresis of resulting fragments from PCR with different DNA templates.** Primers used were plex\_2-forward + plex\_400-reverse.Expected fragment size is 467 bp. Ladder: Thermo Fisher GeneRuler Low Range DNA Ladder

Figure 5 shows the DNA amplified by PCR with the different templates. It can be seen, that the previous version of our domesticated vector (lane 2) is missing base pairs, which seem to be present in the PCR amplification with the other template. Hence, we used this fragment for further ligation attempts.

Unfortunately, we could not completely solve the problem, as sequencing revealed that there were still about 30 bp missing from the plasmid. Thus, a new strategy was employed, relying on cutting out the fragment covering the problematic region from the original pLEXSY\_I-blecherry3 and inserting it into the otherwise domesticated vector.

In order not to re-introduce any BsaI recognition sites, only a specific set of restriction enzymes could be used for this. One of them was ClaI, which is blocked by dam-methylation. In order to bypass this issue, the relevant DNA fragments were transformed into CGSC5127, a *dam- E. coli* strain, followed by plasmid miniprep and digest with ClaI and SpeI.

![]( https://static.igem.org/mediawiki/2021/e/ee/T--TU_Kaiserslautern--Results_domestication_5.jpg)

**Restriction digest of pLEXSY\_I-blecherry3 (1-4) and weird\_plex-old (5-9) with ClaI and SpeI.** Both are shown undigested, respective single digests and double digests. Ladder: Thermo Fisher GeneRuler DNA Ladder Mix

Figure 6 shows the result of the digest with ClaI and SpeI. Lanes 4 and 8 show the expected band patterns of the double digest. Bands where extracted from the gel for further experiments. The ensuing ligation brought out a correctly assembled plasmid, which could be verified by restriction digest as well as sequencing (Fig. 3B) to be the successfully domesticated and intact *weird\_plex* vector.

Another important step in establishing the expression vector for our MoClo system was the introduction of a *lacZ-alpha* gene cassette into the plasmid, which allows for blue white screening after cloning and transformation into *LacZ-omega* carrying *E. coli* cells. Along with the lacZ-α gene cassette, new BsaI restriction sites were introduced into the plasmids that serve as entry site for MoClo assemblies.

![]( https://static.igem.org/mediawiki/2021/d/d1/T--TU_Kaiserslautern--Results_domestication_6.jpg)

**Agarose gel electrophoresis of resulting fragment from PCR on pICH47742 for amplification of the *lacZ-alpha* gene cassette.** Expected fragment size is 623 bp. Ladder: Thermo Fisher GeneRuler DNA Ladder Mix

Figure 7 shows the successful amplification of the *lacZ-alpha* gene cassette originating from pICH47742 (plasmid backbone from the Weber collection)[[1]](#ref-1). After amplification, the *lacZ* fragment as well as the domesticated destination vector were sequentially digested with restriction enzymes BglII and NotI and ligated with T4 DNA ligase, leading to the finished and ready-to-use expression vector weird\_plex.

![]( https://static.igem.org/mediawiki/2021/f/fc/T--TU_Kaiserslautern--Results_domestication_7.jpg)

**Restriction digest of expression vector weird\_plex using SacI.**  Comparison of original vector pLEXSY\_I-blecherry3 to domesticated vector weird\_plex. Ladder: Thermo Fisher GeneRuler DNA Ladder Mix

Figure 8 shows a test digest of the original vector pLEXSY\_I-blecherry3 compared to the domesticated expression vector weird\_plex. The digest along with sequencing of the whole plasmid show definitive proof of a fully assembled and domesticated expression vector containing all important features for our MoClo System in *Leishmania tarentolae.*

## Cloning of parts

To build a library of modular parts, we wanted to design a number of different fluorophores, affinity tags and signal peptides. Eventually, we had to cut down the number of parts, since the domestication of our destination vector turned out to be more time consuming than expected. [This](https://2021.igem.org/Team%3ATU_Kaiserslautern/Parts "This")is our final list of constructs.

Some of the parts we have were acquired via *de novo* synthesis, where we optimized the codon usage for *Leishmania*. [Here](https://2021.igem.org/Team%3ATU_Kaiserslautern/HowToMoClo) you can find out how to create a MoClo part. Some parts were made via PCR using the MoClo collection for *Chlamydomonas reinhardtii* as templates.

![](https://static.igem.org/mediawiki/parts/2/21/T--TU_Kaiserslautern--Engineering_sAP_PCRNEW.png)

Gel electrophoresis of PCR fragment
**1** | PCR *sAP-forward* + *sAP-reverse* on *pLEXSY\_I-blecherry3*
**L** | Thermofisher LowRange GeneRuler [bp]

Our sAP secretion signal was accquired using PCR on the pLEXSY\_I-blecherry3, which is our original vector. Successful PCR amplificates were cloned into the respective target vector using the BbsI restriction sites. The MoClo constructs where then transformed into *E. coli* and selected via blue-white screening.

All acquired L0 parts were test digested and sequenced to confirm the correct insertion of the part into the vector. Correct plasmids were propagated in TOP10 *E. coli* and isolated with plasmid extraction.

![](https://static.igem.org/mediawiki/2021/b/b0/T--TU_Kaiserslautern--Results_L0_test_digests.jpg)

a) Testdigest of all L0-parts in B2, B3 and B4. b) Testdigest of all L0-parts in B3-B4 and B5.
The constructs which are digested with BsaI are marked in green and the ones which are digested with BstEII and SapI are marked in red.

For creating the L1 constructs, we performed overnight MoClo reactions with BsaI and transformed the resulting plasmids in TOP10 *E. coli*. Vectors with an insert were identified via blue-white screening.
Plasmids were propagated in *E. coli* and test digested:

![](https://static.igem.org/mediawiki/2021/3/34/T--TU_Kaiserslautern--Results_L1_digest.jpg )

**Testdigest with SacI of the original pLEXSY\_I-bleCherry\_3, weird plex and various L1-constructs.**

Upon successful assembly of our desired gene construct, the plasmid was almost ready for transfection into *Leishmania*.

The insertion into the *Leishmania* genome works better if the plasmid is linearized before transfection. We achieved this through digesting the plasmid with SwaI, which cuts the plasmid twice to remove the *E. coli* replication cassette (1957 bp).

![]( https://static.igem.org/mediawiki/2021/d/d1/T--TU_Kaiserslautern--SwaI_digests.jpg
)

**SwaI digest of L1 constructs.** Visible are the *E. coli* replication cassette (1957 bp) and the *Leishmania* cassette (5753 bp). Bands run a little higher because a large amount of DNA was loaded.

To increase transfection efficiency of *Leishmania*, a large amount of plasmid DNA (about 5 µg) is needed. Since we lose a little bit of DNA during every step, we linearized 10 µg of DNA using SwaI. The *Leishmania* cassette was extracted from the gel and handed over to the *Leishmania* team.

## Transfection of Leishmania

To finally transfect our level-1 constructs into *Leishmania*, we transfected usually 108 cells with 5-10 µg DNA using a nucleofector. After transfection, the cells were transferred to BHI medium, incubated over night at 27 °C and plated onto BHI agar containing the selection antibiotic bleomycin.

The BHI agar plates were incubated at 27 °C until the colonies reached a diameter of one to two millimeters, which were usually visible after one to two weeks. The colonies were then transferred into liquid medium and induced with tetracycline after three to four days of growth time.

To ensure that the integration of our plasmid into the genome was successful, we performed a colony PCR using primers binding to the blecherry gene.

![](https://static.igem.org/mediawiki/2021/a/a0/T--TU_Kaiserslautern--Agarosegelf%C3%BCrBleomycinresistenz.jpg)

**Agarose gel for bleomycin resistance** (a) Gel electrophoresis for the detection of the blecherry marker. Loaded were respectively M (Marker, Quick-Load Purple 100 bp DNA Ladder) LEXSY original cell culture, pLEXSY\_I-blecherry3 and a negative control Lt UCAS pTB007 without the blecherry marker.

The second lane was loaded with the reaction mixture containing the pLEXSY\_I-blecherry3 culture. The transfected construct carries the blecherry gene and therefore a band at 349 bp should be detectable on the agarose gel. As seen on Figure 13 you can see the agarose gel, one band in the pLEXSY\_I-blecherry3 (Fig.13) construct was visible at the expected size of approximately 340 bp.

Since previous transfection had grown cultures on the negative control, we wanted to make sure that our LEXSY original cell culture did not carry bleomycin resistance.

However, since no band was seen on the agarose gel in the original LEXSY cell culture (Fig. 13), it can be assumed that the Blecherry marker entered the *Leishmania* through transfection and that our transfections are accordingly successful.

The second negative control, Lt UCAS pTB007, was used because it was a culture transfected with a different vector that did not contain the blecherry marker. This way we could be sure that our primer binds to the correct sequence and prove that our transfection was successful.

#### Detection of mCherry production in Leishmania tarentolae

![](https://static.igem.org/mediawiki/2021/f/f1/T--TU_Kaiserslautern--cherry1.jpg)

![](https://static.igem.org/mediawiki/2021/9/9e/T--TU_Kaiserslautern--cherry2.jpg)

![](https://static.igem.org/mediawiki/2021/f/fe/T--TU_Kaiserslautern--cherry3.jpg)

![](https://static.igem.org/mediawiki/2021/2/2e/T--TU_Kaiserslautern--cherry4.jpg)

**Emission and extinction measurement of mCherry.**
Emission (580 nm - 670 nm) and extinction (510 nm - 608 nm) measurements of mCherry using the CLARIOstar plate reader and a 96-well plate. Cells in each cell culture sample were counted and the amount of culture required for 106 cells was calculated. The respective cell culture volume was centrifuged off, the pellet resuspended, and a total volume of 100 µl was pipetted into a well. Triplicates were prepared from each sample. **A)** Spectrum measurement of three tetracycline induced cultures (L1\_3xHA\_RBD\_Strep8His, L1\_sAP\_RBD\_Strep8His and pLEXSY\_I\_blecherry3), one uninduced culture (L1\_sAP\_Myc\_RBD\_Strep8His) and one untransfected *L. tarentolae* cell culture sample as negative control. An mCherry concentration series diluted in BHI medium (1:1000, 1:1500, 1:1200) served as a positive control. To exclude background noise, the spectra of all BHI media used for cultivation was also measured. **B)** Spectrum measurement of three tetracycline induced cultures (L1\_sAP\_RBD\_HA8His, L1\_sAP\_RBD\_Strep8His and L1\_3xHA\_RBD\_Strep8His), one uninduced culture (L1\_sAP\_RBD\_HA8His) and one untransformed *L. tarentolae* cell culture sample as negative control. An mCherry concentration series diluted in BHI medium (1:1000, 1:1500) served as positive control. To exclude background noise, the spectra of all BHI media used for cultivation was also measured.

To detect the production of the protein mCherry, which is produced in fusion with the bleomycin binding protein and encoded by the expression vector, the spectrum of the fluorescent protein was measured in cells harboring different constructs. mCherry has its emission maximum at a wavelength of 610 nm and its extinction maximum at 587 nm [[4]](#ref-4). We also measured uninduced cultures in order to analyze the effect of induction with tetracycline. The addition of tetracycline leads to binding of the tetracycline to the Tet-repressor and thus detachment of the repressor from the operator. Hence expression of mCherry should be lower in uninduced cultures compared to induced cultures [[5].](#ref-5) Cells containing pLEXSY\_I\_blecherry3 (**Fig.14A**) and L1\_sAP\_RBD\_3xHA (induced) (**Fig.14B**) have the highest emission (A: 348909 RFU, B: 345258 RFU) and extinction values (A: 315642 RFU, B: 356666 RFU). The uninduced cultures can not be compared with the induced cultures because they are not originating from the same colony. Hence it is possible that the uninduced cultures show higher expression of proteins than the induced cultures, as protein expression is variable between cultures. Expectedly, the negative controls and the BHI media do not show peaks at the maxima of both spectra. Nevertheless we can verify a successful transfection and the production of mCherry.

## Protein detection

To demonstrate the functionality of our system, we needed to prove that our target proteins were produced by *Leishmania*. Therefore, a big step in our project was detecting the target proteins after transfection using Western blots.
For all blots, we withdrew cell culture containing 108 cells and separated them from the supernatant. The cells were immediately prepared with 5x Laemmli loading buffer diluted to a 1x concentration with 1x cOmpleteTM protease inhibitor including 2% 2-Mercaptoethanol, whereas the supernatant was precipitated with a final concentration of 10% trichloroacetic acid (TCA) and washed with 1 ml acetone priorly. The finished samples were loaded onto 15% SDS gels.

![](https://static.igem.org/mediawiki/2021/3/30/T--TU_Kaiserslautern--results1foto.jpg)

![](https://static.igem.org/mediawiki/2021/b/bc/T--TU_Kaiserslautern--results2foto.jpg)

**Immunoblots stained with RBD antibodies.**

As a negative control (n.c.) we used the cell pellet (P) and supernatant (S) of either LEXSY original cell culture **(A)**, culture containing our destination vector weird plex **(C, D)** or both **(B)**. RBD-GFP served as positive control (p.c.). All induced (+) and uninduced cultures on one blot descended from the exact same colony to show the actual difference caused by tetracycline. Lanes marked with x were left empty.

All blots show a significant increase in the amount of protein after the induction with tetracycline. Furthermore, it can be observed that the supernatant contains far more protein in all samples including sAP constructs and the RBD is cleaved off leading two double bands, whereas cells only show one band and constructs without sAP do not show bands in the supernatant as seen in **15A** and **15B**. To further validate the results we achieved with the RBD antibodies, we stained blots with HIS and GST antibodies.

![](https://static.igem.org/mediawiki/2021/e/e3/T--TU_Kaiserslautern--idc.jpg)

**Immunoblots stained with HIS (A) and GST (B) antibodies.**

Weird plex was used as a negative control, Subs4-His8 **(A)** and GST **(B)** as positive controls. The membranes were stained with anti HIS and anti GST. Lanes marked with x were left empty.

Although both blots do not show a gain in protein amount comparing uninduced cells and supernatant, we can still see this in the induced samples, as well as a distinct difference between induced and uninduced cultures.

Most of the bands lay higher than expected, which suggests an underlying technical problem, for example the percentage of the SDS gels at 15% being too high. But as the heights add up in relation to each other, we are still convinced that the results are valid, especially because we were able to confirm them with different antibodies.

## Protein purification

Protein purification showed itself to be more difficult than we expected. After a lot of trials and errors, purifications using different affinity tags, trying a lot of different protocols, we finally were able to get some results.

![](https://static.igem.org/mediawiki/2021/e/e8/T--TU_Kaiserslautern--GST_blots.jpeg )

**Purification of GST-tagged RBD with Glutathione-Sepharose affinity chromatography**. 50 mL of supernatant of *L. tarentolae* cultures transfected with L1\_sAP\_RBD\_GST (38.44\*106 cells/ml), were concentrated to 2 mL using a Centricon with 10 kDa cut-off and loaded onto a Glutathion-sepharose column. Samples taken from flow-through, wash and four eluate fractions. A mix of all eluate fractions was precipitated with TCA (Eluate TCA). Samples were analysed using SDS-PAGE and Western blot. The blot was stained with an antibody against the RBD. MW – molecular weight marker. a) RBD staining. a) GST staining

To make use of recombinant proteins in medicine, research, or therapeutics you need to be able to purify the protein. We tried purifying our RBD from *Leishmania* supernatant via a Glutathion-Sepharose column, to which our GST-tagged RBD binds. The Western blot showed a clear signal for the GST-RBD (51.1 kDa) in our TCA precipitated eluate. Also, there seems to be RBD (25.1 kDa) in the washing steps, from which we concluded that our GST tag gets cleaved off to some extent.

We stained the same blot again with GST to confirm this (Figure 17.b). The signal for free GST was far stronger than that for the RBD-GST fusion protein (Figure 17.a). The purification process seemed to work, although it looks like we lost a lot of GST during the washing steps. The precipitated eluate also showed a RBD-GST signal (51.5kDa) as well as GST only (26.5 kDa). Therefore, we confirmed that the GST tag is partially cleaved off at the TEV cleavage site, which is located between the RBD and the GST.

![]( https://static.igem.org/mediawiki/2021/0/03/T--TU_Kaiserslautern--Results_BCA_Assay.jpg )

**BCA-Assay of purified RBD-GST**. Eluate TCA (Figure 17) was used to determine protein concentration.

The BCA assay resulted in a concentration of 0.13 mg/ml protein in the eluate. Which means in our total of 3 mL eluate we had 0.4 mg of our RBD-GST which comes down to 7.6 µg of protein in 1 mL of culture.

The GST purification left us with GST-tagged RBD. To purify only RBD we made use of the TEV cleavage site in between the RBD and the GST tag.

![]( https://static.igem.org/mediawiki/2021/5/5b/T--TU_Kaiserslautern--GST_HIS_Blots.jpg )

**Cleaving at the TEV site and purification of RBD**. The eluate with purified RBD-GST was digested with TEV-protease to cleave off the purification tag. Another GST purification was carried out to remove free GST. The RBD should be in the flow-through and wash fractions, while GST should remain on column. The TEV protease contains a His-tag to remove it after digestion via nickel affinity chromatography. Flow-through and wash fractions were applied to a nickel affinity column. The flow-through should contain the pure RBD.

Apparently, the concentration of the RBD-GST fusion protein was too low to detect via Western blotting and only a faint band (51.1 kDa) was visible in input and eluate of after GST purification. The purification of GST was successful, although much GST remained in flow-through and wash fractions, although the amount of column material was calculated for the protein concentration that was determined via the BCA assay (Figure 18).

![]( https://static.igem.org/mediawiki/2021/c/c4/T--TU_Kaiserslautern--Another_GST.jpeg )

**Purification of GST-tagged RBD with Glutathione-Sepharose affinity chromatography**. 50 mL of supernatant of *L. tarentolae* cultures transfected with L1\_sAP\_RBD\_GST (55.31\*106 cells/ml), were concentrated to 2 mL using ammonium sulphate precipitation. The protein pellet was resuspended in PBS and loaded onto a Glutathion-Sepharose column. Samples taken from flow-through, wash and two eluate fractions were analysed. A mix of all eluate fractions was precipitated with TCA (Eluate TCA). As a negative control, weird\_plex lysate and supernatant were used. After resuspending the protein pellet in PBS, a sample was centrifuged to obtain supernatant (P. Supernatant) and pellet. GST positive control (GST-p.c., 26.4 kDa) and RBD positive control (RBD-GFP, 53.3 kDa) served to ensure the functionality of the antibodies used. Samples were analysed using SDS-PAGE and Western blotting. The blot was stained against GST. MW – molecular weight marker.

The purification shows large amounts of GST as well as of the RBD-GST in all fractions. P. supernatant and P. resuspended both show RBD-GST (51.5 kDa) as well as GST (26.4 kDa), indicating that not all of our protein was soluble after precipitation with ammonium sulphate.

![](https://static.igem.org/mediawiki/2021/3/3d/T--TU_Kaiserslautern--mCerul_mVenus_blots.jpg )

**Purification of RBD\_mCerulean\_GST and RBD\_mVenus\_GST with Glutathione-Sepharose affinity chromatography**. 50 mL of supernatant of L. tarentolae cultures transfected with L1\_sAP\_RBD\_GST (38\*106 cells/ml), were concentrated to 2 mL using a Centricon with 10 kDa cut-off. The concentrate was loaded onto a Glutathion-Sepharose column. Samples were taken from input, flow-through, wash and two eluate fractions. A mix of all eluates was precipitated with TCA (Eluate TCA). Samples were analysed using SDS-PAGE and Western blotting. The membrane was stained with an antibody against GST. MW – molecular weight marker. A) RBD\_mCerulean\_GST B) RBD\_mVenus\_GST.

We wanted to purify our RBD\_mCerulean/mVenus using GST affinity chromatography to finally carry out our activity assay with RBD, where we could show the binding of the RBD to the ACE2 receptor via fluorescence of the fused mCerulean or mVenus. The signal of GST is visible in input, flow-through, wash 1, as well as in all eluate fractions. The eluate fractions show three signals. The first one (78.2 kDa) represents the entire fusion protein. All bands seem to run with a slightly larger apparent MW than expected, which could be due to glycosylations. The second band resembles mCerulean-GST/mVenus-GST. Here it appears that the RBD was cleaved off, pointing to presence of a protease that cleaves between the RBD and the fluorophore. The third band (26.4 kDa) represents free GST, which indicates another cleavage at the TEV recognition site.

## Activity assay

Not only did we want to show that our protein gets expressed in the supernatant, or that it can successfully be purified through a GST-column, but that it also is functional. For this we chose two different methods. The activity of the RBD can be proven via Western blot, and with the right tag, through fluorescence microscopy.

For this method, we used human HEK 293T +ACE2 +TMPRSS2 (HEK+) cells that we got from our sponsor VectorBuilder. This specific cell line has a mutation that ensures an overexpression of ACE2 (angiotensin I converting enzyme 2), which is the protein on the surface of the human cells, where the RBD binds. As negative control we used HEK 293T cells that don’t express ACE2 (HEK-).

We applied the maximum volume that we had at our disposal and incubated the HEK-cells with our protein for an hour at 37°C and with 5% CO2. Following the incubation, we removed the medium and washed the cells with PBS and with that, all the residuals away. If our RBD successfully bound to the ACE2 protein on the cell membrane of the HEK-cells, it should have stuck, even after washing. Prior to loading the samples into SDS-gel wells, we lysed the cells with RIPA Buffer. On the blot we used primary antibodies against RBD (m α RBD 1:2000) and separately against ACE2 (m α ACE2 1:1000) with which we detected the specific proteins.

We incubated one well of HEK-cells with RBD\_Strep8His and another well with weird plex (w\_plex), where we only concentrated the supernatant from *Leishmania* through ammonium sulfate precipitation. One further well HEK-cells were incubated with RBD\_TEV\_GST which was purified through an GST column. All those samples were also incubated with HEK--cells that did not express ACE2 as negative controls.

![](https://static.igem.org/mediawiki/2021/4/4d/T--TU_Kaiserslautern--vorletzerblot.jpg)

**Immunoblot stained with antibodies against ACE2 and RBD.** The samples were loaded on an 10% SDS-Gel. As a marker, Pageruler Prestained Plus was used. We loaded 20 µL from each sample. **A.** 1: HEK-- cells incubated with 200 µL RBD\_TEV\_GST which was purified, 2: HEK+-cells incubated with 1.5 mL RBD\_Strep8His which was concentrated through ammonium sulfate precipitation, 3: HEK+-cells incubated with 1.5 mL w\_plex, to show that there is no cross reaction with any proteins in *Leishmania* supernatant, 4: HEK+- cells incubated with 200 µL RBD\_TEV\_GST which was purified, 5: HEK--cells incubated with 1.5 mL RBD\_Strep8His with was concentrated through ammonium sulfate precipitation, 6: HEK+-cells only, 7: HEK-cells only. The blot was incubated with m α ACE2 (1:1000, Santa Cruz) overnight. **B.** Here the blot was treated with m α RBD (1:2000, R&W).

For the protein RBD\_Strep8His on HEK+ cells we can see a band at the right height, which is a first indication of functionality of our protein RBD. Furthermore, the band is not present in the negative control (HEK--RBD-Strep8His) where we incubated the HEK--cells with the same sample, which proves that the band is not a cross reaction of the antibody with any other proteins of the HEK-cells or *Leishmania* supernatant.

The sample with RBD\_TEV\_GST shows no such band. This could be because the RBD got lost during purification, or because the GST tag, which is as big as the RBD itself, interferes with the binding with ACE2. The HEK+-cells that were incubated with w­\_plex show, as expected, no RBD.

The following blot (Figure 23) was treated the same way as the one mentioned above (Figure 22), but with a few additional constructs. None of the added constructs showed a band at the expected molecular weight from RBD, but the potential activity of the sample RBD\_Strep8His could be replicated with another set of HEK+-cells (Figure 23).

![](https://static.igem.org/mediawiki/2021/c/ca/T--TU_Kaiserslautern--letzterblot.jpg)

**Second immunoblot with antibodies against ACE2 and RBD.** The HEK + and HEK--cells were incubated with 250 µL sample except for RBD\_Strep8His and w\_plex where 1.5 mL were loaded. The samples were loaded on an 10% SDS-Gel. As a marker, Pageruler Prestained Plus was used. We loaded 20 µL from each sample. RBD\_TEV\_GST A – E were samples of the same construct, but through different steps or treated differently. If a purification occurred prior to incubation with HEK +-cells, it is always with an GST column. 1: A) 500 mL of *L. tarentolae* supernatant were concentrated through amicons with a cutoff of 10.000, and afterwards purificated. 2: B) It is the previous sample but followed by an incubation with TEV-Protease. 3: C) 50 ml of *L. tarentolae* were concentrated through amicons with a cutoff of 10.000, followed by a purification and treated with TEV-protease. 4: D) 50 ml of *L. tarentolae* supernatant were concentrated through ammonium sulfate precipitation, with subsequent purification 5: E) The sample contained the same sample as the previous one, but with an additional step of treatment with TEV-protease. 6: For RBD\_Strep8His 50 ml of *L. tarentolae*. supernatant were concentrated through ammonium sulfate precipitation. 7: 50 mL of *L. tarentolae* supernatant, were w\_plex was transfected, was concentrated through ammonium sulfate precipitation.

The band by the RBD\_Strep8His sample is clearly visible, and no cross contamination of the antibodies could be detected in the negative control where no protein should be in the supernatant (w\_plex) or nothing was incubated (HEK+). In addition to that, the RBD-GFP construct could be detected as a positive control for the specific binding of the antibody (Fig.23).

In Addition to the western blots, we looked at incubated HEK-cells with samples of RBD with a fluorescent tag through the microscope. The only difference to the cell preparation for blots, is an embedded coverglass in the 6-well-plate prior to sowing the cells. To visualize the nucleus, it was stained with DAPI solution added to a solution that extends the emission of the fluorophores (ProLong™ Glass Antifade Mountant).

The microscopy images (Figure 24) were from HEK+ and HEK--cells, incubated with RBD\_mCerulean\_TEV\_GST that was purified through an GST column.

![](https://static.igem.org/mediawiki/2021/4/43/T--TU_Kaiserslautern--mikroskopie.png)

**Fluorescence microscopy of HEK-cells with RBD\_mCerulean\_TEV\_GST.** The HEK+and the HEK--cells were incubated for one hour with 250 µL of purificated RBD\_mCerulean\_TEV\_GST in 37°C and 5% CO2. Prior to the microscopy, they were treated with DAPI solution to stain the nucleus. **A-C.** HEK--cells with DAPI stain and incubation with RBD\_mCerulean\_TEV\_GST. **D-F.** HEK+-cells with DAPI stain and incubation with RBD\_mCerulean\_TEV\_GST.

It is clearly noticeable that the HEK+-cells are mutants and therefore not as fit as the HEK--cells. The mCerulean fluorophore has the same excitation wavelength as DAPI, which makes it difficult to distinguish them from one another. Nevertheless, a slight shimmer around the HEK+-cells is visible, that is not apparent in the HEK--cells. That could be an indication for the binding of RBD onto the ACE2 on the cell membrane.

We also had another fluorophore tag mVenus, and incubated the sample RBD\_mVenus with HEK- - cells and with HEK+ - cells (Fig. 25) The images showed ambiguous results. We can see yellow structures at certain areas at the cell membrane, but those are not very clear. This maybe could be a first indication for a further functional protein.

![](https://static.igem.org/mediawiki/2021/8/89/T--TU_Kaiserslautern--letztemicro.jpg)

**HEK+ - cells incubated with RBD\_mVenus.** The HEK+ - cells were treated with RBD\_mVenus. This image was taken on a coverglass with DAPI solution, which also included the ProLong solution.

However, those experiments were only done once (Figure 23), or twice (Figure 24 and 25) and therefore should be considered as preliminary data. To definitively prove the functionality of our RBD, more replicates are needed. This work will be continued in the next months, and till then be hopefully ready for publication. More, and different kinds of activity assays are scheduled, like pull downs and immunoprecipitation, and immunofluorescence.

## Outlook

During the course of the year we were able to build a modular library of genetic parts suitable for *Leishmania* and domesticate the respective destination vector. With this vector and the library, we were able to successfully transfect *L. tarentolae* and detect our desired proteins. Although protein purification was more complicated than expected, we were able to purify the RBD, our protein of interest and even showed its functionality via an activity assay with human ACE2-cells.

For further improvement of the MoClo Mania system, the library has to be expanded, new parts e.g. more fluorophores or detection tags can be added, as well as more different purification tags to offer an even bigger variety of parts.

To improve the expression level of the constructs, there are a variety of possibilities.
Via growth curve and protein quantification we want to find out when after adding tetracycline protein harvesting makes most sense and if its concentration impacts protein yield.

We also want to improve the purification of our protein and try out some more purification methods like HPLC or FPLC. We are also facing the problem that our TEV cleavage site gets cleaved in *Leishmania*, so we have to think about how to solve this problem. One idea was a more specific protease inhibitor mix during growth of *Leishmania*, as [Dr. Christian Janzen](https://2021.igem.org/Team%3ATU_Kaiserslautern/Human_Practices#head-2)suggested, as well as adding different protease inhibitors during purification itself. Another possibility regarding the cleavage of our fusion protein might be changing the TEV site to another cleavage site.
Furthermore, we want to investigate the glycosylation capabilities of *L. tarentolae* using different comparison methods, e.g., mass spectrometry or reversed-phase chromatography.[[6]](#ref-6)

Apart from the improvements on our library and the general project, we also want to further improve and test our example protein, the receptor binding domain (RBD) of Sars-CoV-2. We want to show the binding to human ACE2-receptor again, but via a fluorophore fused to the RBD e.g. L1\_sAP\_RBD\_mVenus\_TEV-GST.
We also want to further research on to the mechanism of the RBD and want to expand our work and not only express the RBD but the full length Sars-CoV-2 S protein. The S protein of the Sars-CoV-2 is a highly glycosylated protein which makes *Leishmania* a suitable expression host. Since the glycosylation pattern of the S protein plays a major role in binding to the human ACE2 receptor and therefore the immune response, our research could provide more insights into a possible protein based vaccine or ELISA based antibody tests.[[7]](#ref-7)

### Sources

1. [**Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S** (2011) A modular cloning system for standardized assembly of multigene constructs, PLoS One 6](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016765)
2. [**Stephan Klatt, Michael Rohe, Kathirvel Alagesan, Daniel Kolarich, Zoltan Konthur, and Daniela Hartl** (2013) Production of Glycosylated Soluble Amyloid Precursor Protein Alpha (sAPPalpha) in Leishmania tarentolae, Proteome Res. 12](https://pubmed.ncbi.nlm.nih.gov/23214446/)
3. <https://www.jenabioscience.com/files/jenabioscience/datasheet_extern/EGE-1410.pdf>
4. <https://www.fpbase.org/protein/mcherry/>
5. [Kushnir S, Gase K, Breitling R, Alexandrov K. Development of an inducible protein expression system based on the protozoan host Leishmania tarentolae. Protein Expr Purif. 2005 Jul;42(1):37-46. doi: 10.1016/j.pep.2005.03.004. Epub 2005 Mar 25. PMID: 15939291.](https://pubmed.ncbi.nlm.nih.gov/15939291/)
6. [**Angelo Palmigiano, Angela Messina, Luisa Sturiale, Domenico Garozzo;** Chapter Six - Advanced LC-MS Methods for N-Glycan Characterization; Comprehensive Analytical Chemistry, Elsevier, Volume 79, 2018, Pages 147-172, https://doi.org/10.1016/bs.coac.2017.06.009](https://www.sciencedirect.com/handbook/comprehensive-analytical-chemistry/vol/79/suppl/C)
7. [**Reis, C. A., Tauber, R., & Blanchard, V. (2021).** Glycosylation is a key in SARS-CoV-2 infection. Journal of molecular medicine (Berlin, Germany), 99(8), 1023–1031. https://doi.org/10.1007/s00109-021-02092-0](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8140746/)

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