iGEM 2019 — Team Stuttgart

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# Team:Stuttgart/Notebook

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Team

[Team Members](https://2019.igem.org/Team%3AStuttgart/Team)
[Collaborations](https://2019.igem.org/Team%3AStuttgart/Collaborations)
[Sponsoring](https://2019.igem.org/Team%3AStuttgart/Sponsoring)

Project

[Description](https://2019.igem.org/Team%3AStuttgart/Description)
[Design](https://2019.igem.org/Team%3AStuttgart/Design)
[Experiments](https://2019.igem.org/Team%3AStuttgart/Experiments)
[Notebook](https://2019.igem.org/Team%3AStuttgart/Notebook)
[Contribution](https://2019.igem.org/Team%3AStuttgart/Contribution)
[Results](https://2019.igem.org/Team%3AStuttgart/Results)
[Demonstrate](https://2019.igem.org/Team%3AStuttgart/Demonstrate)
[Improve](https://2019.igem.org/Team%3AStuttgart/Improve)
[Attributions](https://2019.igem.org/Team%3AStuttgart/Attributions)

Parts

[Parts Overview](https://2019.igem.org/Team%3AStuttgart/Parts)
[Basic Parts](https://2019.igem.org/Team%3AStuttgart/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AStuttgart/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AStuttgart/Part_Collection)

Human Practices

[Human Practices](https://2019.igem.org/Team%3AStuttgart/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AStuttgart/Public_Engagement)

[Safety](https://2019.igem.org/Team%3AStuttgart/Safety)

Awards

[Hardware](https://2019.igem.org/Team%3AStuttgart/Hardware)
[Measurement](https://2019.igem.org/Team%3AStuttgart/Measurement)
[Model](https://2019.igem.org/Team%3AStuttgart/Model)
[Software](https://2019.igem.org/Team%3AStuttgart/Software)

[Judging Form](https://igem.org/2019_Judging_Form?team=Stuttgart)

## Project

# Notebook

* Additional work
* Dry lab
* Vibrio
* Algae

| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week 17 04/22/2019-04/28/2019 | Preperation for iGEM Spring Festival/ Meeting with Analytic-Jena AG | Benedikt Schober/ Katharina Hofer, Jan Seeger, Sini Münßinger | Posterdesign and introduction video./ Presentation of the iGEM team and our project and preliminary discussion for the use of a loaned qPCR tower for the analyses in the field of tRNA quantification. |  |
| Week 18 04/29/2019-05/05/2019 | iGEM Spring Festival | Katharina Kocher, Kai Schülke, Marvin Bubeck, Benedikt Schober | We took part in the european meetup in Bonn and presented our project for the first time. |  |
| Week 19 05/06/2019-05/12/2019 | Meeting with Analytik-Jena AG | Katharina Hofer, Jan Seeger, Sini Münßinger | Second preliminary discussion for the use of a loaned qPCR tower for the analyses in the field of tRNA quantification. |  |
| Week 20 05/13/2019-05/19/2019 | Meeting with Promega Corporation | Katharina Hofer, Sini Münßinger, Lars Höing | Presentation of the iGEM team and our project in relation to potential assays and molecular biology analysis methodologies that we might be provided with. In addition, preliminary discussion of a qPCR seminar in Tübingen, which was cancelled at short notice. |  |
| Week 22 05/27/2019-06/02/2019 | Visit Symbiosis and GeneBridges/ Meeting with Analytik-Jena AG | Sini Münßinger, Jan Seeger, Jannis Stadager, Verena Haug / Katharina Hofer, Jan Seeger, Sini Münßinger | We presented the companies Symbiosis and GeneBridges our project and got valuable inputs. / Third preliminary discussion for the use of a loaned qPCR tower for the analyses in the field of tRNA quantification. |  |
| Week 25 06/17/2019-06/23/2019 | Meeting with Eurofins Genomics | Sini Münßinger, Jan Seeger, Jannis Stadager, Verena Haug | We presented our team and project and discussed the sequencing that would probably be necessary. |  |
| Week 27 07/01/2019-07/07/2019 | German iGEM Meetup / Meeting with Analytik-Jena AG | Benedikt Schober, Liv Paul, Luca Schelle, Jan Notheisen, Philip Horz / Katharina Hofer, Jan Seeger, Sini Münßinger | We presented our project in Düsseldorf at the German Meetup resulting in collaborations with serveral german iGEM Teams. / Transport of the qPCR device provided on loan with subsequent introduction. |  |
| Week 28 07/08/2019-07/14/2019 | Meeting with Infors HT | Jan Seeger | The project was presented and we talked about the cultivation of vibrio and algae. |  |
| Week 31 07/29/2019-08/04/2019 | Developing a questionare for local political parties in regards to the fears and gains of synthetic biology | Jannis Stadager | In order to gain an understanding about the opinion on synthetic biologie we developed a questionaire about gains and fears of synthetic biology as well as our project. The answers we got back lead to the participation on the pupil fair in Karlsruhe in order to explain synthetic biology to the public |  |
| Week 36 09/02/2019-09/08/2019 | Meet the managing dircetor of Genaxxon | Sini Münßinger, Jan Seeger | Mr. Tröndle provided us GreenMasterMIx and Hotscriptase for the qPCR experiments. During the meeting he gave us useful tips concerning our experiments. |  |
| Week 37 09/09/2019-09/15/2019 |  |  |  |  |
| Week 38 09/16/2019-09/22/2019 |  |  |  |  |
| Week 39 09/23/2019-09/29/2019 | Representing the iGEM Stuttgart Team at a exhibition for graduating pupils in Karlsruhe. qPCR team visited Dispendix and recieved support for the preparation of dilution experiments. | Jannis Stadager, Dimitri Graf, Sini Münßinger, Jan Seeger, Hannah Berreth, Corinna Kersten |  |  |
| Week 40 09/30/2019-10/06/2019 | Meeting with Micro-Biolytics | Sini Münßinger, Dimitri Graf, Jannis Stadager, Benedikt Schober | We talked with the CEO of Micro-Biolytics Andreas Wolf about bioanalytical possibilities concerning the measurement and the dynamics of the media composition. |  |

| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week 14 04/01/2019-04/07/2019 | Modelling of gene expression in Vibrio natriegens | Jan Müller | A gene expression simulation of GFP and GFP derivatives in Vibrio natriegens was run. We could successfully validate, that a rare codon tag slows down the protein translation initially and that this burden can be overcome by increasing the rare tRNA availability. | [Model](https://2019.igem.org/Team%3AStuttgart/Model) |
| Week 15 04/08/2019-04/21/2019 | Modelling of gene expression in Vibrio natriegens | Jan Müller | A gene expression simulation of GFP and GFP derivatives in Vibrio natriegens was run. We could successfully validate, that a rare codon tag slows down the protein translation initially and that this burden can be overcome by increasing the rare tRNA availability. | [Model](https://2019.igem.org/Team%3AStuttgart/Model) |
| Week 16 04/15/2019-04/21/2019 | Vibrio natriegens' codon usage | Jan Müller | Evaluation of Vibrio natriegens' codon usage and identification of rare tRNAs. | [Design](https://2019.igem.org/Team%3AStuttgart/Design) |
|  | Modelling of proposed airlift photobioreactor design | Jannis Stadager, Dimitri Graf | A CFD Simulation based on the preliminary specs of the airlift photobioreactor was run. The goal was to verify the airlift characteristica even after addition of light tubes into the reactor | [Model](https://2019.igem.org/Team%3AStuttgart/Model) |
| Week 17 04/22/2019-04/28/2019 | Design of the ptRNA Plasmid | Jan Müller |  | [Design](https://2019.igem.org/Team%3AStuttgart/Design) |
|  | Modelling of proposed airlift photobioreactor design | Jannis Stadager, Dimitri Graf | A CFD Simulation based on the preliminary specs of the airlift photobioreactor was run. The goal was to verify the airlift characteristica even after addition of light tubes into the reactor | [Model](https://2019.igem.org/Team%3AStuttgart/Model) |
| Week 18 04/29/2019-05/05/2019 | Modelling of proposed airlift photobioreactor design | Jannis Stadager, Dimitri Graf | A CFD Simulation based on the preliminary specs of the airlift photobioreactor was run. The goal was to verify the airlift characteristica even after addition of light tubes into the reactor | [Model](https://2019.igem.org/Team%3AStuttgart/Model) |

![](https://static.igem.org/mediawiki/2019/7/74/T--Stuttgart--logo-vibrio.png)

# *Vibrio*

Vibrio

* [Wet lab](#wetlab)
* [Biobrik](#biobrik)
* [qPCR](#qPCR)

## Vibrio - Wet lab

| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week 23 06/03/2019-06/09/2019 | ptRNA\_backbone -Ligation | Benedikt Schober | The linearized fragment from IDT was ligated using the T4-Ligase. Afterwards the ligated ptRNA-backbone was transformed in E. coli Dh5a cells. | https://static.igem.org/mediawiki/2019/a/af/T--Stuttgart--ptRNA\_backbone.png |
| Week 24 06/10/2019-06/16/2019 | Amplifaction of tRNA-Fragments | Kai Schülke ,Jan Müller | The tRNA fragments were synthetized by IDT and amplified by PCR. | The PCR was sucessful. The DNA was checked and purified via agarose gel electrophoresis. |
| Week 25 06/17/2019-06/23/2019 | competent cells Vibrio natriegens DSM759 | Benedikt Schober, Katharina Kocher | competent Vibrio natriegens DSM 759 were made according to iGEM Marburg 2018 | Competent vibrio natriegens DSM 759 cells were checked on performance via transformation of the sfGFP-Vector. |
| Week 26 06/24/2019-06/30/2019 | competent cells Escherichia coli DH5a | Kai Schülke, Benedikt Schober, Katharina Kocher | chemically and electro-competent Escherichia coli DH5a cells were made. | Competent escherichia coli cells were checked on performance via transformation of a pUC19a Vector. |
| Week 27 07/01/2019-07/07/2019 | growth curve V.natriegens in BHIN | Jan Müller, Jan Seeger, Katharina Kocher | Growth curves of the new Vibrio natriegens DSM 759 in BHIN without plasmid. | The resulting growth curve is shown below. |
| Week 28 07/08/2019-07/14/2019 | Cloning of tRNA fragments into ptRNA\_backbone | Katharina Kocher, Benedikt Schober | The ptRNA\_backbone and the tRNA fragments AGA, AGG, CGG, TGC, TCC and the combined tRNA fragment were digested using the restriction enzymes XbaI and SpeI. After purification the digested fragments were ligated using the T4 DNA ligase. | Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA\_backbone. |
| Week 29 07/15/2019-07/21/2019 | Cloning of tRNA fragments into ptRNA\_backbone | Katharina Kocher, Benedikt Schober | The ptRNA\_backbone and the tRNA fragments AGA, AGG, CGG, TGC, TCC and the combined tRNA fragment were digested using the restriction enzymes PstI and EcoRI. After purification the digested fragments were ligated using the T4 DNA ligase. | Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA\_backbone. |
| Week 30 07/22/2019-07/28/2019 | Plasmid-Preperation, Agarose-Gel, Agarose-Gel-Extraction, Clean and Concentrator Kit | Katharina Kocher, Benedikt Schober | The ptRNA\_backbone and the tRNA fragments AGA, AGG, CGG, TGC, TCC and the combined tRNA fragment were digested using the restriction enzymes PstI and EcoRI. After purification the digested fragments were ligated using the T4 DNA ligase. | Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA\_backbone. |
| Week 31 07/29/2019-08/04/2019 | Cloning of tRNA fragements into pSB1C3 | Katharina Kocher; Benedikt Schober | The vector pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC, TCC and the combined tRNA fragment were digested using the restriction enzymes XbaI and SpeI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning. | Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only pSB1C3. |
| Week 32 08/05/2019-08/11/2019 | Cloning of tRNA fragments into pSB1C3 | Katharina Kocher; Benedikt Schober | The vector pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC, TCC and the combined tRNA fragment were digested using the restriction enzymes XbaI and SpeI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning. | Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only pSB1C3. |
| Week 33 08/12/2019-08/18/2019 | Cloning of tRNA fragments into pSB1C3 | Katharina Kocher; Benedikt Schober | The vector pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC, TCC and the combined tRNA fragment were digested using the restriction enzymes EcoRI and PstI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning. | Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only pSB1C3. |
| Week 34 08/19/2019-08/25/2019 | Cloning of tRNA fragments into pSB1C3 | Katharina Kocher; Benedikt Schober | The vector pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC, TCC and the combined tRNA fragment were digested using the restriction enzymes EcoRI and PstI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning. | Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only pSB1C3. |
| Week 35 08/26/2019-09/01/2019 | Cloning of tRNA fragments into pSB1C3 | Katharina Kocher; Benedikt Schober | After a re-synthesis of the ptRNA\_backbone, the tRNA fragments AGA, AGG, CGG, TGC, TCC,the combined tRNA fragment and the pTRNA\_backbone were digested using the restriction enzymes EcoRI and PstI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning. | Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA\_backbone. |
| Week 36 09/02/2019-09/08/2019 | Cloning of tRNA fragments into ptRNA\_backbone | Katharina Kocher; Benedikt Schober | After a re-synthesis of the ptRNA\_backbone, the tRNA fragments AGA, AGG, CGG, TGC, TCC,the combined tRNA fragment and the pTRNA\_backbone were digested using the restriction enzymes EcoRI and PstI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning. | Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA\_backbone. |
| Week 37 09/09/2019-09/15/2019 | Cloning of tRNA fragments into ptRNA\_backbone | Katharina Kocher; Benedikt Schober | After a re-synthesis of the ptRNA\_backbone, the tRNA fragments AGA, AGG, CGG, TGC, TCC,the combined tRNA fragment and the pTRNA\_backbone were digested using the restriction enzymes EcoRI and PstI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning. | Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA\_backbone. |
| Week 38 09/16/2019-09/22/2019 | Cloning of tRNA fragments into ptRNA\_backbone | Katharina Kocher; Benedikt Schober | After a re-synthesis of the ptRNA\_backbone, the tRNA fragments AGA, AGG, CGG, TGC, TCC,the combined tRNA fragment and the pTRNA\_backbone were digested using the restriction enzymes EcoRI and PstI. After purification the digested fragments were ligated using the T4 DNA ligase. Following transformation into DH5α revealed no successful cloning. | Following transformation into DH5α revealed no successful cloning. The colonies obtained showed no insert in an agarose gel and only ptRNA\_backbone. |
| Week 39 09/23/2019-09/29/2019 | gDNA Isolation | Benedikt Schober | The tRNA fragments AGA, AGG, CGG, TGC, TCC were amplified from the Vibrio natriegens DSM 759 genome chr.1. | The PCR was succesfully checked by agarose gel electrophoresis. |
| Week 40 09/30/2019-10/06/2019 | Mutagenisis of sfGFP, improvement of Gfp and sfGfp biobricks for vibrio natriegens | Katharina Kocher, Sabrina Holl, Hannah Berreth, Verena Haug | Tags of rare Codons (AGA, AGG, CGG, TGC, TCC) were introduced into the sfGFP vector using PCR. The sfGFP with tags of rare codons was transformed into DH5α. Inserts with sfGFP and GFP were introduced into vector backbone pSB1C3. | Following transformation into DH5α revealed no successful cloning. No colonies were obtained after transformation. Following transformation of pSB1C3 containing genetic information of sfGFP or GFP into vibrio natriegens revealed no colonies. |
| Week 41 10/07/2019-10/13/2019 | Improvement of Gfp and sfGfp biobricks for vibrio natriegens | Hannah Berreth, Verena Haug | Inserts with sfGFP and GFP were introduced into vector backbone pSB1C3. | Following transformation into vibrio natriegens revealed no colonies. |
| Week 42 10/14/2019-10/20/2019 | Improvement of Gfp and sfGfp biobricks for vibrio natriegens | Hannah Berreth, Verena Haug | Inserts with sfGFP and GFP were introduced into vector backbone pSB1C3. | Following transformation into vibrio natriegens revealed no colonies. |
|  | Expression of improved GFP and sfGFP in Vibrio natriegens | Marvin Bubeck, Jan Müller, Hannah Berreth, Verena Haug | Inserts with sfGFP and GFP were introduced into vector backbone pSB1C3. | Following transformation into vibrio natriegens revealed no colonies. |
|  | Cloning of tRNA fragments into ptRNA\_backbone | Katharina Kocher; Sabrina Holl | Cloning of the tRNA fragments into the ptRNA\_backbone was performed according to the NEBuilder HiFi DNA Assembly protocol. | Following transformation into DH5α revealed no successful cloning. No colonies were obtained. |
|  | Transformation of pRARE plasmid in Vibrio natriegens and growth curves | Katharina Kocher; Sabrina Holl | The pRARE plasmid was prepared from E.coli Rosetta and transformed into Vibrio natriegens. Growth curves of the wild type Vibrio natriegens and Vibrio natriegens cells with pRARE were performed. | Growth curves reveald no differences in growth velocity between wild type Vibrio natriegens and Vibrio natriegens cells with pRARE. |
|  | Transformation of sfGFP containing tags of rare codons into Vibrio natriegens. | Katharina Kocher; Sabrina Holl | Tags of rare Codons (AGA, AGG, CGG, TGC, TCC) were introduced into the sfGFP vector using PCR. The sfGFP with tags of rare codons was transformed into Vibrio natriegens. | Following transformation into Vibrio natriegens revealed no successful cloning. No colonies were obtained after transformation. |

## Vibrio - Biobrik

| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week 40 09/30/2019-10/06/2019 | Biobrick charakterisation | Sini Münßinger, Katharina Hofer, Jan Seeger | Plamsid DNA was transformed in E. coli MG1655 via electroporation. |  |
| Week 41 10/07/2019-10/13/2019 | Biobrick charakterisation | Sini Münßinger, Katharina Hofer, Jan Seeger | For the characterisation, E. coli was cultivated and the RNA purificated by Zymo Quick RNA. During the cultivation, mRFP fluorescence was measured. qPCR was performed to quantify the mRNA amount of mRFP. |  |
| Week 42 10/14/2019-10/20/2019 | Biobrick charakterisation | Sini Münßinger, Katharina Hofer, Jan Seeger |  |  |

## Vibrio - qPCR

| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week 26 06/24/2019-06/30/2019 | Vibrio natriegens glycerol stocks | Katharina Hofer | Based on a lyophilized Vibrio natriegens pellet, glycerol stocks were produced. |  |
| Week 32 08/05/2019-08/11/2019 | RNA purification of Vibrio natriegens wildtyp | Sini Münßinger, Katharina Hofer, Jan Seeger | Comparison of two different RNA purification kits: Zymo Quick RNA (Zymo Research) and Nucleospin miRNA (Macherey & Nagel; kit is able to purificate small RNA (>200nt). |  |
| Week 33 08/12/2019-08/18/2019 | First execution of whole protocol | Sini Münßinger, Katharina Hofer, Jan Seeger | The whole protocol was carried out for the first time. RNA purificated by both kits was used. qPCR was performed with KAPA mastermix. qPCR with the cDNA based on the M&N RNA was performed again, PCR products were sequenzed by Eurofins. | Melting curve showed several amplification products based on the RNA of Zymo kit. RNA purificated by M&N kit will used for further experiments because the meltling curve showd only one amplification product. Each tRNA species showed different ct-values. PCR products were loaded on a gel, but the amplificates were shorter than the expected length. Results of sequencing showed no amplification of the complete sequence (adapter and tRNA). |
| Week 34 08/19/2019-08/25/2019 | qPCR with GreenMasterMix | Sini Münßinger, Katharina Hofer, Jan Seeger | Ligation and reverse transcrition were performed. GreenMasterMix (Genaxxon) was used for the qPCR. qPCR products were sequenzed again. | Each tRNA species showed different ct-values. PCR products were loaded on a gel, but the amplificates were shorter than the expected length. Results of sequencing showed no amplification of the complete sequence (adapter and tRNA). |
| Week 35 08/26/2019-09/01/2019 | qPCR with GreenMasterMix | Sini Münßinger, Katharina Hofer, Jan Seeger | Ligation and reverse transcrition were performed. GreenMasterMix (Genaxxon) was used for the qPCR. | Each tRNA species showed different ct-values. PCR products were loaded on a gel, but the amplificates were shorter than the expected length. |
| Week 36 09/02/2019-09/08/2019 | Comparison of tRNA concentration after different times of cultivation | Sini Münßinger, Katharina Hofer, Jan Seeger | Vibrio was cultivated as usual, samples were taken after 1,5h (mid exponential phase) and 3h (beginning stationary phase). RNA purification and protocol as usual. In addition purified E.coli tRNA (Merck) was treated as usual to verify the protocol. | No differences between the samples of 1,5h and 3h regarding the ct-values. PCR products were loaded on a gel, but the amplificates were shorter than the expected length. |
| Week 37 09/09/2019-09/15/2019 | Comparison of tRNA concentration after different times of cultivation | Sini Münßinger, Katharina Hofer, Jan Seeger | Vibrio was cultivated as usual, samples were taken after 1,5h (mid exponential phase) and 3h (begin stationary phase). RNA purification and protocol as usual. In addition purified E.coli tRNA (Merck) was treated as usual to verify the protocol. | No differences between the samples of 1,5h and 3h regarding the ct-values. PCR products were loaded on a gel, but the amplificates were shorter than the expected length. |
| Week 38 09/16/2019-09/22/2019 |  | Sini Münßinger, Katharina Hofer, Jan Seeger |  |  |
| Week 39 09/23/2019-09/29/2019 | Dilution experiment, preparation of E. coli experiment | Sini Münßinger, Katharina Hofer, Jan Seeger | Verification of the possibilty to determine different tRNA concentrations. Purified V. natriegens tRNA was diluted 1:1, 1:10, 1:100. Sample preparation and qPCR protocol as usual. E. coli Bl21 DE3 and E. coli Bl21 DE3 pRARE were cultivated in 2YT-medium. Cells were harvested after 2,5h, 3,5h and 5,5h and stored at -70°C. | Differences between tRNA concentrations are detectable by ct-value. |
| Week 40 09/30/2019-10/06/2019 | Dilution experiment, comparison of Ecoli BL21 DE3 and pRARE | Sini Münßinger, Katharina Hofer, Jan Seeger | Diltion experiment was performend with all tRNA species in triplicates. Samples were pipetted by Dispendix. Comparison of tRNA concentration of Ecoli BL21 DE3 and BL21 DE3 pRARE. | Differences between tRNA concentrations are detectable by ct-value. No differences between E. coli BL21 DE3 and BL21 DE3 pRARE. |
| Week 40 09/30/2019-10/06/2019 | Implementation of Hotscriptase, | Sini Münßinger, Katharina Hofer, Jan Seeger | Because of the implementation of Hotscriptase (Genaxxon) a new protocol was developed. Treatment of all tRNA species of V. natriegens and Ecoli BL21 DE3 and BL21 DE3 pRARE with new Hotscriptase protocol. | Each tRNA species showed different ct-values. PCR products were loaded on a gel, but the amplificates were shorter than the expected length. qPCR showed different ct-values for the tRNA species of E. coli BL21 DE3 and BL21 DE3 pRARE. The results obtained were not as expected. |
| Week 41 10/07/2019-10/13/2019 | Cultivation and tRNA quantification of E. coli Rosetta | Sini Münßinger, Katharina Hofer, Jan Seeger | tRNA concentation of E. coli BL21 DE3 wildtype, pRARE and Rosetta was quantified and compared via qPCR. | qPCR showed different ct-values for the tRNA species of E. coli BL21 DE3, BL21 DE3 pRARE and Rosetta. The results obtained were not as expected. |

![](https://static.igem.org/mediawiki/2019/6/60/T--Stuttgart--logo-alge.png)

# *Algae*

Algae

* [Medium and Growth](#medium)
* [Cultivation](#cultivation)
* [Disruption](#disruption)

## Algae - Medium development, analysis and growth experiments

| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week 32 08/05/2019-08/11/2019 | first experiments growing bacteria on media based on chlorella vulgaris | Verena Haug, Hannah Berreth | E.coli was grown on medium based on either algae or yeast without trypton. Various salt concentratrion were tested. |  |
| Week 35 08/26/2019-09/01/2019 | HPLC Analysis of the Amino Acid composition of the first algae extract | Jannis Stadager, Dimitri Graf | The algae extract without enrichment was analyzed for all 20 amino acids using a HPLC | Only 5 amino acids were able to be detected. It was concluded, that the medium has to be enriched for cultivation of bacteria |
| Week 38 09/16/2019-09/22/2019 | Growth curve on vibrio | Marvin Bubeck, Corinna Kersten | The Algae extract was mixed with two concentrations of additional NaCl and as an alternative supplemented with soy peptone. As control, variations of LB and BHIN mediums where produced in two concentrations with NaCl. V. natriegens was cultivated in all medium variants at 37°C. | V. natriegens grew on all mediums. |
| Week 39 09/23/2019-09/29/2019 | HPLC Analysis of the Amino Acid composition of the algae extract | Jannis Stadager, Dimitri Graf | The algae extract was solved in water (30 g/L and 60 g/L) having the same concentration as when cultivating bacteria on the algae extract. A amino acid HPLC was performed with measuring all 20 amino acids | Not all 20 amino acids were able to be detected. This was caused by a malfunction in the HPLC measurement which was seen in the high variation of the control standard (GABA) |
| Week 40 09/30/2019-10/06/2019 | Growth curve of E. coli and V. natriegens on PhyCoVi medium | Marvin Bubeck | The Algae extract was mixed with two concentrations of additional NaCl and as an alternative supplemented with soy peptone. As control, variations of LB and BHIN mediums where produced in two concentrations with NaCl. E. coli and V. natriegens were cultivated in all medium variants at 37°C. | Both bacteria grew on all mediums. |
| Week 41 10/07/2019-10/13/2019 | HPLC Analysis of the carbonhydrate compostion of the algae extract. Startpoint of sampling the airlift reactor for our human practices partner Microbiolytics | Jannis Stadager, Dimitri Graf | The algae extract was solved in water (30 g/L) having the same concentration as when cultivating bacteria on the algae extract. A sugar HPLC was performed with measuring Glucose, Fructose, Raffinose as well as Sucrose. | A large amount of Glucose was able to be detected with the other sugars hardly being in a detectable range |
| Week 42 10/14/2019-10/20/2019 | Growth curve of B. subtillis and C. glutamicum on PhyCoVi medium | Marvin Bubeck, Dimitri Graf, Jannis Stadager | The Algae extract was mixed with two concentrations of additional NaCl and as an alternative supplemented with soy peptone. As control, variations of LB and BHIN mediums where produced in two concentrations with NaCl. B. subtillis was cultivated in all medium variants at 37°C, C. glutamicum was cultivated in all medium variants at 30°C. | Both bacteria grew on all mediums. |

## Algae - Cultvation

| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week 14 04/01/2019-04/07/2019 | Start cultivation of chlorella vulgaris in shaking flasks | Jannis Stadager, Verena Haug, Dimitri Graf, Hannah Berreth | Chlorella vulgaris was seeded in 300 mL shaking flasks containing DSN media | Measurement of OD750 showed growth of Chlorella vulgaris in DSN medium. |
| Week 15 04/08/2019-04/21/2019 | Planning of various cultivation methods | Hannah Berreth, Verena Haug, Jannis Stadager, Dimitri Graf, Marvin Bubeck, Sabrina Holl, Corinna Kersten | Self built 10 L bioreactors as well as other bioreactor designs were discussed and planned. Strategies for faster biomass generation including stirring, gassing and ilumination were discussed. | Building of 10 L bioreactor with red and blue lighting via LED as well as gassing with a pump and stirring with a magnetic stirrer was decided. |
| Week 18 04/29/2019-05/05/2019 | Start cultivation of chlorella vulgaris in up to 10 l self-built bioreactors, start cultivation of chlorella sorokineana in shaking flasks, Modelling of airlift bioreactor design | Jannis Stadager, Dimitri Graf, Hannah, Verena | Inoculation of large-scale lab bioreactor with chlorella vulgaris. | Faster growth rate than in shaking flasks was observed. |
| Week 24 06/10/2019-06/16/2019 | Planning of airlift bioreactor design | Jan Notheisen, Luca Schelle, Philip Horz |  | Meeting with Prof. Takors provided us with several ideas for the airlift design |
| Week 25 06/17/2019-06/23/2019 | Planning of airlift bioreactor design | Jan Notheisen, Luca Schelle, Philip Horz |  | Further research helped filter which design and method is achievable and realizable in the short timeframe |
| Week 26 06/24/2019-06/30/2019 | OD CDW correlation of Chlorella vulgaris, Planning of airlift bioreactor design | Jannis Stadager, Dimitri Graf, Jan Notheisen, Luca Schelle, Philip Horz | Chlorella vulgaris culture was stepwise diluted; OD and CDW were measured and correlated. | We were able to obtain a CDW-OD correlation, that was used for the estimation of the amount of algae in our bioreactor. |
| Week 27 07/01/2019-07/07/2019 | Building of 37 l airlift bioreactor | Jan Notheisen, Luca Schelle, Philip Horz |  | Reactor was succesfully set up |
| Week 28 07/08/2019-07/14/2019 | Start cultivation of chlorella vulgaris in 37 l airlift bioreactor | Jan Notheisen, Luca Schelle, Philip Horz, Verena Haug, Jannis Stadager, Dimitri Graf | Inoculation of self-built airlift bioreactor with inoculum of 10 l bioreactor with Chlorella vulgaris. | Faster growthrate was observed in 37 L airlift bioreactor than in 10 L bioreactor |
| Week 29 07/15/2019-07/21/2019 | Start of long-term OD-CDW correlation, first experiments growing bacteria on media based on chlorella vulgaris | Verena Haug, Hannah Berreth, Jannis Stadager, Dimitri Grafi, Marvin Bubeck, Sabrina Holl, Corinna Kersten | Duplicates of 10 mL sapmles were taken daily from the airlift bioreacor. OD and CDW were measured and correlated. |  |
| Week 30 07/22/2019-07/28/2019 | HPLC-Analysis | Sabrina Holl, Corinna Kersten | Sugar HPCL of algea cell extraction with autolysis. | Highest amounts of sugar were seen with autolysis at pH3. |
| Week 31 07/29/2019-08/04/2019 | rFAN-Assay Establishing | Dimitri Graf | General protocol for the rFan assay, for fast estimation of free amino acids. |  |
| Week 32 08/05/2019-08/11/2019 | rFAN-Assay, autolysis | Corinna Kersten | Testing autolysis as a method for cell wall disruption combined with bead-milling and quantifying amino acids with rFAN-Assay | Yeast served as positiv control. It revealed the highest amount of free amino acids in relation to the used biomass. The yield was higher at pH12. C. vulgaris and C. Sorokiniana reached a precentage of free amino acids around 0.05 %. |
| Week 33 08/12/2019-08/18/2019 | Quantifiying Cell wall disruption by Anthrone assay and Bradford assay | Corinna Kersten, Marvin Bubeck |  |  |
| Week 35 08/26/2019-09/01/2019 | End of long term OD-CDW correlation | Hannah Berreth, Verena Haug, Jannis Stadager, Dimitri Graf, Marvin, Sabrina, Corinna | Duplicates of 10 mL sapmles were taken daily from the airlift bioreacor. OD and CDW were measured and correlated. | We were not able to obtain a clear trend out of our measurements. The correlation was discarded. |
| Week 38 09/16/2019-09/22/2019 | Start cultivation of chlorella sorokineana in 37 l airlift bioreactor | Jan Notheisen, Luca Schelle, Philip Horz, Dimitri Graf | Inoculation of self-built airlift bioreactor with inoculum of 10 l bioreactor with Chlorella sorokiniana. | Not only Chlorella vulgaris but also Chlorella sorokiniana had a better groth rate in 37 L airlift bioreactor than in 10 L bioreactor. |

## Algae - Disruption

| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week | Experiment | Participents | Description | Results |
| --- | --- | --- | --- | --- |
| Week 20 05/13/2019-05/19/2019 | Cell extraction tests using ulrasound and ball mills | Hannah Berreth, Dimitri Graf | 5 mL algae (OD750=2) were used as sample for cell extraction. Various sonification times and shaking times in the ball mill were tested and samples were taken for FACS analysis. | Extract was frozen and measured at week 23 |
| Week 21 05/20/2019-05/26/2019 | Cell extraction tests using high pressure homogenizer and autoclav | Verena Haug, Jannis Stadager | 5 mL algae (OD750=2) were used as sample for cell extraction. Various repeats of high pressure homigenizing were performed. Samples were autoklaved as well. Samples were taken for FACS analysis. | Extract was frozen and measured at week 23 |
| Week 22 05/27/2019-06/02/2019 | Cell extraction tests using acids and base as well as mortar | Sabrina Holl, Marvin Bubeck | 5 mL algae (OD750=2) were used as sample for cell extraction. Various acid and base concentration were used various mortar strategies were tested. Samples were taken for FACS analysis. | Extract was frozen and measured at week 23 |
| Week 23 06/03/2019-06/09/2019 | FACS analysis of extracted samples | Jannis, Verena Haug, Hannah Berreth | FACS analysis was performed with samples of cell extraction methods. Hereby we measured a sample with the same OD and Volume as used for the extraction and set the total count as 100%. The efficiency of the cell disruption was then calculated based on the referenz to 100%. | In general mechanical disruption (stirred ball mill, mixer) of microalgae proved to be more efficient than chemical or heatinduced |
| Week 24 06/10/2019-06/16/2019 | Cell extraction tests using autolysis and ball mill | Jannis Stadager, Verena Haug | Cell autolysis and ball mill were used as extraction methods. Methods were also combined. | Algae were treated and incubated at different timepoints, analyzation was done in week 26 |
| Week 25 06/17/2019-06/23/2019 | Cell extraction tests using autolysis and ball mill | Jannis Stadager, Verena Haug | Cell autolysis and ball mill were used as extraction methods. Methods were also combined. | Algae were treated and incubated at different timepoints, analyzation was done in week 26 |
| Week 26 06/24/2019-06/30/2019 | Cell extraction tests using autolysis and ball mill | Jannis Stadager, Verena Haug | Cell autolysis and ball mill were used as extraction methods. Methods were also combined. Analysis was performed via Bradford assays and Anthron-assays to detect proteins and sugars. | In general, a combination of acid autolysis at a high temperature with a subsequent ball mill proved to reult in a higher level of sugars and proteins |
| Week 33 08/12/2019-08/18/2019 | Cell extraction | Marvin Bubeck, Corinna Kersten | Cell extraction was tested with different amounts and sizes of beads for bead-milling. The extraction was quantified with Bradford-Assay and Anthron-Assay. | An amount of 80% beads with a size between 200-300 µm led to the highest yield in protein. 50 % beads led to much lower yield of protein. |
| Week 34 08/19/2019-08/25/2019 | Cell extraction | Marvin Bubeck, Corinna Kersten | Cell-extraction was tested with boiling the algea in 1 M KOH, 5 M KOH, 1 M NaOH or 1 M HCl. The extraction was quantified with Bradford-Assay and Anthron-Assay | Highest amounts of Protein was achieved by 5 M KOH. Lowest protein yield was seen with 1 M NaOH. 1 M KOH and 1 M HCl led to smiliar amounts of protein. |


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# Team:Stuttgart/Experiments

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[Parts Overview](https://2019.igem.org/Team%3AStuttgart/Parts)
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[Part Collection](https://2019.igem.org/Team%3AStuttgart/Part_Collection)

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[Human Practices](https://2019.igem.org/Team%3AStuttgart/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AStuttgart/Public_Engagement)

[Safety](https://2019.igem.org/Team%3AStuttgart/Safety)

Awards

[Hardware](https://2019.igem.org/Team%3AStuttgart/Hardware)
[Measurement](https://2019.igem.org/Team%3AStuttgart/Measurement)
[Model](https://2019.igem.org/Team%3AStuttgart/Model)
[Software](https://2019.igem.org/Team%3AStuttgart/Software)

[Judging Form](https://igem.org/2019_Judging_Form?team=Stuttgart)

## Project

# Experiments

| Name | Category | Group |
| --- | --- | --- |
| [OD CDW\_correlation\_of\_Chlorella\_vulgaris\_by\_dilution](https://static.igem.org/mediawiki/2019/6/68/T--Stuttgart--Protocol_OD-CDW_correlation_of_Chlorella_vulgaris_by_dilution.pdf) | Cultivation | Algae |
| [OD-CDW\_correlation\_of\_algae\_Chlorella\_vulgaris](https://static.igem.org/mediawiki/2019/4/4d/T--Stuttgart--Protocol_OD-CDW_correlation_of_algae_Chlorella_vulgaris.pdf) | Cultivation | Algae |
| [Media first experiments](https://static.igem.org/mediawiki/2019/5/55/T--Stuttgart--Protocol_media_first_experiments.pdf) | Medium Analysis | Algae |
| [HPLC](https://static.igem.org/mediawiki/2019/9/9f/T--Stuttgart--Protocol_HPLC.pdf) | Medium Analysis | Algae |
| [rFAN\_Assay](https://static.igem.org/mediawiki/2019/8/83/T--Stuttgart--Protocol_rFAN_Assay.pdf) | Algae extraction | Algae |
| [Algae Extraction](https://static.igem.org/mediawiki/2019/e/ec/T--Stuttgart--Protocol_Algae_Extraction.pdf) | Algae extraction | Algae |
| [Cell extraction with autolysis combined with bead-milling](https://static.igem.org/mediawiki/2019/e/ec/T--Stuttgart--Protocol_Cell_extraction_with_autolysis_combined_with_bead-milling.pdf) | Algae extraction | Algae |
| [Chlorella Vulgaris first extraction methods](https://static.igem.org/mediawiki/2019/f/f3/T--Stuttgart--Protocol_Chlorella_Vulgaris_first_extraction_methods.pdf) | Algae extraction | Algae |
| [Anthrone Assay](https://static.igem.org/mediawiki/2019/f/f3/T--Stuttgart--Experiments_AnthroneAssay.pdf) | Algae extraction | Algae |
| [BradfordAssay](https://2019.igem.org/File%3AT--Stuttgart--Experiments_BradfordAssay.pdf) | Algae extraction | Algae |

| Name | Category | Group |
| --- | --- | --- |
| [Plasmid Preparation](https://static.igem.org/mediawiki/2019/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf) | Molecular Biology | Vibrio |
| [BioBrick Cloning](https://static.igem.org/mediawiki/2019/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf) | Molecular Biology | Vibrio |
| [Gibson Assembly](https://static.igem.org/mediawiki/2019/6/6c/T--Stuttgart--Protocol_Gibson_Assembly.pdf) | Molecular Biology | Vibrio |
| [Competent Cells](https://static.igem.org/mediawiki/2019/4/41/T--Stuttgart--Protocol_Competent_Cells.pdf) | Molecular Biology | Vibrio |
| [Transformation](https://static.igem.org/mediawiki/2019/8/83/T--Stuttgart--Protocol_Transformation.pdf) | Molecular Biology | Vibrio |
| [Gel Extraction](https://static.igem.org/mediawiki/2019/3/32/T--Stuttgart--Protocol_Gel_Extraction.pdf) | Molecular Biology | Vibrio |
| [Agarose Gel](https://static.igem.org/mediawiki/2019/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf) | Molecular Biology | Vibrio |
| [Clean and Concentrate](https://static.igem.org/mediawiki/2019/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf) | Molecular Biology | Vibrio |
| [NEBuilder HiFi DNA Assembly](https://static.igem.org/mediawiki/2019/2/25/T--Stuttgart--Protocols_NEBuilder_HiFi_DNA_Assembly.pdf) | Molecular Biology | Vibrio |
| [PCR](https://static.igem.org/mediawiki/2019/f/f4/T--Stuttgart--Protocol_PCR.pdf) | Molecular Biology | Vibrio |
| [gDNA Extraction](https://static.igem.org/mediawiki/2019/c/c0/T--Stuttgart--gDNA_extraction.pdf) | Molecular Biology | Vibrio |
| [Q5 site-directed Mutagenesis](https://static.igem.org/mediawiki/2019/e/ed/T--Stuttgart--Q5_Site-directed_mutagenesis.pdf) | Molecular Biology | Vibrio |
| [Blunt end ligation](https://static.igem.org/mediawiki/2019/2/2e/T--Stuttgart--Blunt_End_Ligation.pdf) | Molecular Biology | Vibrio |
| [Deacylierung and precipitation of tRNA](https://static.igem.org/mediawiki/2019/f/f3/T--Stuttgart--Deacetylation_and_precipitation_of_tRNA.pdf) | qPCR | Vibrio |
| [Hybridization and ligation of tRNAs](https://static.igem.org/mediawiki/2019/a/a8/T--Stuttgart--Hybridization_and_ligation_of_tRNAs.pdf) | qPCR | Vibrio |
| [Reverse transcription of the specific tRNA species](https://static.igem.org/mediawiki/2019/9/9f/T--Stuttgart--Reverse_transcription_of_the_specific_tRNA_species.pdf) | qPCR | Vibrio |
| [RT qPCR without prior execution of a reverse transcription](https://static.igem.org/mediawiki/2019/8/85/T--Stuttgart--RT_qPCR_without_prior_execution_of_a_reverse_transcription.pdf) | qPCR | Vibrio |
| [RT qPCR](https://static.igem.org/mediawiki/2019/7/73/T--Stuttgart--RT_qPCR.pdf) | qPCR | Vibrio |
| [Purification of RNA smaller than 200 nucleotides](https://static.igem.org/mediawiki/2019/3/3d/T--Stuttgart--Purification_of_RNA_smaller_than_200_nucleotides.pdf) | qPCR | Vibrio |
| [Growth Curve Vibrio Natriegens](https://static.igem.org/mediawiki/2019/f/f0/T--Stuttgart--Protocol_Growth_Curve_Vibrio_natriegens.pdf) | Cultivation | Vibrio |
| [BHI-Agar](https://static.igem.org/mediawiki/2019/3/37/T--Stuttgart--Protocols_BHI-Agar.pdf) | Cultivation | Vibrio |
| [LB-Agar](https://static.igem.org/mediawiki/2019/1/12/T--Stuttgart--Protocols_LB-Agar.pdf) | Cultivation | Vibrio |
| [2YT medium](https://static.igem.org/mediawiki/2019/5/57/T--Stuttgart--Protocol_2YT_medium.pdf) | Cultivation | Vibrio |
| [Glycerol stock Vibrio](https://static.igem.org/mediawiki/2019/0/03/T--Stuttgart--Protocol_Glycerol_stock_Vibrio_natriegens.pdf) | Cultivation | Vibrio |
| [Cultivation Vibrio natriegens for RNA purification](https://static.igem.org/mediawiki/2019/c/c0/T--Stuttgart--Protocol_Cultivation_Vibrio_natriegens_for_RNA_purification.pdf) | Cultivation | Vibrio |
| [Cultivation Escherichia coli for RNA purification](https://static.igem.org/mediawiki/2019/6/64/T--Stuttgart--Protocol_Cultivation_Escherichia_coli_for_RNA_purification.pdf) | Cultivation | Vibrio |

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# Team:Stuttgart/Results

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[Home](https://2019.igem.org/Team%3AStuttgart)

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[Team Members](https://2019.igem.org/Team%3AStuttgart/Team)
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Project

[Description](https://2019.igem.org/Team%3AStuttgart/Description)
[Design](https://2019.igem.org/Team%3AStuttgart/Design)
[Experiments](https://2019.igem.org/Team%3AStuttgart/Experiments)
[Notebook](https://2019.igem.org/Team%3AStuttgart/Notebook)
[Contribution](https://2019.igem.org/Team%3AStuttgart/Contribution)
[Results](https://2019.igem.org/Team%3AStuttgart/Results)
[Demonstrate](https://2019.igem.org/Team%3AStuttgart/Demonstrate)
[Improve](https://2019.igem.org/Team%3AStuttgart/Improve)
[Attributions](https://2019.igem.org/Team%3AStuttgart/Attributions)

Parts

[Parts Overview](https://2019.igem.org/Team%3AStuttgart/Parts)
[Basic Parts](https://2019.igem.org/Team%3AStuttgart/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AStuttgart/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AStuttgart/Part_Collection)

Human Practices

[Human Practices](https://2019.igem.org/Team%3AStuttgart/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AStuttgart/Public_Engagement)

[Safety](https://2019.igem.org/Team%3AStuttgart/Safety)

Awards

[Hardware](https://2019.igem.org/Team%3AStuttgart/Hardware)
[Measurement](https://2019.igem.org/Team%3AStuttgart/Measurement)
[Model](https://2019.igem.org/Team%3AStuttgart/Model)
[Software](https://2019.igem.org/Team%3AStuttgart/Software)

[Judging Form](https://igem.org/2019_Judging_Form?team=Stuttgart)

## Project

# Results

Vibrio

* [qPCR](#qPCR)
* [Growth curves *Vibrio natriegens*](#growthVibrio)
* [Cloning of tRNA fragments](#cloningtRNA)
* [Tansformation of pRARE into Vibrio natriegens](#transformationpRARE)
* [Mutagenesis of sfGFP](#mutagenesis)

Algae

* [Media based on algae](#algae-media-based)
* [Autolysis in combination
  with bead-milling Results](#autolysis)
* [Cell wall disruption of *C. vulgaris* and *C. sorokiniana*](#celldisruption)
* [CDW correlation](#cdwcorrelation)
* [CDW-OD correlation by dilution](#cdwodcorrelation)
* [Preliminary Cell Disruption](#preliminarycelldisruption)
* [Growth Curves of various bacteria on PhyCoVi Medium](#growthCurvesOnBacteria)
* [Sugar HPLC Results](#sugarHPLCResults)

## qRT-PCR for the relative quantification of specific tRNA-species

Alongside with the generation of a climate-friendly medium, the goal of our project PhyCoVi was to optimize
the strain
*Vibrio natriegens* for a potential use in the biotech industry. The optimization is performed on the
genomic level to increase the intracellular availability of tRNA species. As a result, the strain’s
performance to express heterologous proteins is enhanced.

A method needs to be developed to quantify individual tRNA species specifically to prove the increased
expression not only on the protein level.  Multiple methods can be found to quantify non-coding RNA
1, 2 or total tRNA concentration 3, 4. Whereas finding a well-established method to
quantify single tRNA species specifically is in vain. The only method paper was published in the journal
“RNA biology” in 2015 by Honda *et al.*: “Four-leaf clover qRT-PCR: A convenient
method for selective quantification of mature tRNA” 5. The authors of this paper removed
the amino acid at the 3’ end followed by hybridization and ligation with a DNA/RNA hybrid stem loop
creating a “four-leaf clover” shaped appearance of the tRNA ligation product. The stem loop
adaptor contained a TaqMan probe binding site. During the qPCR the TaqMan probe was cut by exonuclease
function of the used polymerase resulting in emission of fluorescence.

Building on the work of Honda *et al.* we developed a new and simplified method for relative
quantification of specific tRNA species without the necessity of TaqMan probes. Instead using a DNA/RNA
hybrid stem loop we used a linear DNA/RNA construct as adaptor.

The first step is to isolate RNA with a length of < 200 nt from cultured *V. natriegens* cells.
Then, the amino acid bound to the 3’ end needs to be removed by a deacylation reaction. This results
in a sticky end, where a linear RNA/DNA hybrid adaptor can be ligated, which is complementary to the
3’ end overhang. Although different tRNAs show differences in length and sequence, the last three
nucleotides at the 3’ end are the same for all tRNA species. The ligated adaptor contains a binding
site for the forward-primer, which is identical for all tRNAs (unspecific primer). We used T4-RNA-ligase 2
that requires ATP. For this reason, a polynucleotide kinase was necessary to carry out a phosphorylation
reaction at the 5’ end.

To amplify single tRNA species specifically, we distinguished between two options. First option was using
the specific tRNA primer in a reverse transcription to convert the whole tRNA pool to cDNA. Following RNase
H digestion results in pure cDNA of the desired tRNA species.

Later the desired tRNA species is amplified during a qPCR by using the specific reverse primer and the
unspecific adaptor primer.

During qPCR a DNA-intercalating fluorescence dye (Green DNA dye) allows for relative quantification: Green
DNA dye binds to double stranded DNA and absorbs blue light and emits green light. The more double stranded
DNA is generated, the higher the resulting fluorescence. And the higher the concentration of the template in
the sample the faster the fluorescence exceeds the threshold. The number of cycles at which this happens is
called the threshold cycle (Ct). (e.g. if sample A showed a Ct of 8 and sample B
showed a Ct of 11, sample A contained 23 = 8 times more template.)

After running a DNA gel, we noticed that the obtained amplification products did not show the expected
length. This may have been a result of distinct secondary structures of the tRNA species: the reverse
transcription reaction was performed at 42 °C which is the enzyme’s optimum working temperature.
However, this temperature is not high enough to prevent secondary structures or to break them up. Therefore,
areas with secondary structures may have been inaccessible for the reverse transcriptase resulting in
shorter cDNA fragments.

For this reason, we tested a second option to amplify single tRNA species specifically. A modified
polymerase together with the specific reverse primer can be used to amplify the desired tRNA species using
RNA as a template. This modified polymerase works at temperatures around 65 °C and can use both RNA and
DNA as a template. The reverse transcription reaction is thus not needed as a consecutive step anymore.
Moreover, the modified enzyme creates the specific cDNA from RNA directly and the high temperature prevents
secondary structures. The relative quantification based on Ct values is the same as in the option
described before.

### References

1. I. A. Babarinde, Y. Li, A. P. Hutchins (2019) Computational Methods for Mapping, Assembly and
   Quantification for Coding and Non-coding Transcripts, Computational and Structural Biotechnology
   Journal, Vol. 17, pp 628-637

2. D. Jacob, K. Thüring, A. Galliot, V. Marchand, A. Galvanin, A. Ciftci, K. Scharmann, M. Stock,
   J.‐Y. Roignant, S.A. Leidel, Y. Motorin, R. Schaffrath, R. Klassen, M. Helm (2019) Absolute
   Quantification of Noncoding RNA by Microscale Thermophoresis, Angewandte Chemie International Edition,
   Vol. 58, pp 9565 – 9569

3. T. S. Stenum, M. A. Sørensen, S. L. Svenningsen (2017) Quantification of the Abundance and
   Charging Levels of Transfer RNAs in *Escherichia coli*. Journal of Visual Experiments,
   Issue 126, e56212

4. Y. Guo, A. Bosompem, S.Mohan, B. Erdogan, F.Ye, K. C. Vickers, Q. Sheng, S. Zhao, C. Li, P.-F. Su, M.
   Jagasia, S. A. Strickland, E. A. Griffiths, A. S. Kim (2015) Transfer RNA detection by small RNA deep
   sequencing and disease association with myelodysplastic syndromes, BMC Genomics, 16:727

5. S. Honda, M. Shigematsu, K. Morichika, A. G. Telonis, Y. Kirino (2015) Four-leaf clover qRT-PCR: A
   convenient method for selective quantification of mature tRNA, RNA Biology, Vol. 12, pp 501 – 508

## tRNA quantification of rare codons in *V. natriegens* with reverse transcription and the use of ‘GreenMasterMix without ROX’

**tRNA quantification of rare codons in *V. natriegens* with reverse transcriptase**

At the beginning different parameters such as the temperatures during the qPCR or primer and RNA
concentrations in the sample preparation were tested. Samples were taken in mid exponential phase of
*V. natriegens*. After several experiments, the results of the optimized protocol are shown in figure
1. Experiments were conducted using the following protocols: [Purification\_of\_RNA\_smaller\_than\_200\_nucleotides.pdf](https://static.igem.org/mediawiki/2019/3/3d/T--Stuttgart--Purification_of_RNA_smaller_than_200_nucleotides.pdf),
[Deacetylation\_and\_precipitation\_of\_tRNA.pdf](https://static.igem.org/mediawiki/2019/f/f3/T--Stuttgart--Deacetylation_and_precipitation_of_tRNA.pdf),
[Reverse\_transcription\_of\_the\_specific\_tRNA\_species.pdf](https://static.igem.org/mediawiki/2019/9/9f/T--Stuttgart--Reverse_transcription_of_the_specific_tRNA_species.pdf),
[Hybridization\_and\_ligation\_of\_tRNAs.pdf](https://static.igem.org/mediawiki/2019/a/a8/T--Stuttgart--Hybridization_and_ligation_of_tRNAs.pdf), [RT\_qPCR.pdf](https://static.igem.org/mediawiki/2019/7/73/T--Stuttgart--RT_qPCR.pdf).

Figure 1: tRNA quantification of V. natriegens with ‘GreenMasterMix without ROX’: fluorescent
intensity over cycle number is shown. AGA is represented by the pink curve, AGG by the dark blue one, CGG is
coloured in light blue, GAA in green. The orange curve shows TCC and red TGC. The black curve shows the
negative control. The experiment was carried out in triplicates, the standard deviation is represented by
the error bars.

The first curve that appears at a Ct value of 12.59 was AGA. AGG showed a Ct value of
21.04. It is followed by GAA with 26.26 and TGC with 27.5. The Ct value of 30.17 belongs to TCC.
The highest Ct value showed CGG with 30.74 cycles.

Figure 2 – 7 show the melting curves of the qPCR products. A single peak indicates that there is only
one amplified product for each codon.

Figure 2: The melting curve of the amplification product of the AGA tRNA in V. natriegens is shown. The
generated product has a melting temperature of about 77.5 ° C.

Figure 3: The melting curve of the amplification product of the AGG tRNA in V. natriegens is shown. The
amplification product has a melting temperature of about 77.5 ° C.

Figure 4: The melting curve of the amplification product of the CGG tRNA in V. natriegens is shown. The
melting temperature of the amplification product was about 77.5 ° C.

Figure 5: The melting curve of the amplification product of the GAA tRNA in V. natriegens is shown. The
generated product has a melting temperature of about 77.5 ° C.

Figure 6: The melting curve of the amplification product of the TCC tRNA in V. natriegens is shown. The
amplified product has a melting temperature of about 77.5 ° C.

Figure 7: The melting curve of the amplification product of the TGC tRNA in V. natriegens is shown. . The
melting temperature of the amplification product was about 77.5 ° C.

After the protocol was established, we wanted to find out if we were able to detect any differences in tRNA
concentration between the exponential growth phase and the stationary phase. Therefore, we cultivated
*V. natriegens* and harvested cells after 1.5 h and 3 h for RNA isolation. Samples were treated as
usual but Ct values for each tRNA species between the sampling times showed no differences (data
not shown).

To verify the RNA purification and the other steps of the protocol, purified and lyophilized
*E. coli* tRNA (Roche, Basel, Switzerland) was treated like the purified *V. natriegens* RNA.
Results of qPCR showed similar curve characteristics and Ct values referring to the results based
on *V. natriegens* (data not shown).

**tRNA quantification of rare codons in *V. natriegens* with HotScriptase**

“HotScriptase RT”, a modified polymerase, is able to perform the reverse transcription of RNA
and the amplification of cDNA in one step. Due to the higher temperatures, tRNA secondary structure should
be reduced, which should lead to a more efficient reverse transcription. ([RT\_qPCR\_without\_prior\_execution\_of\_a\_reverse\_transcription.pdf](https://static.igem.org/mediawiki/2019/8/85/T--Stuttgart--RT_qPCR_without_prior_execution_of_a_reverse_transcription.pdf)). Steps before the reverse transcription and qPCR, were the same as usual (<Purification_of_RNA_smaller_than_200_nucleotides.pdf>, <Deacetylation_and_precipitation_of_tRNA.pdf>,
[Hybridization\_and\_ligation\_of\_tRNAs.pdf](https://static.igem.org/mediawiki/2019/a/a8/T--Stuttgart--Hybridization_and_ligation_of_tRNAs.pdf)).

Figure 8: tRNA quantification of rare codons of V. natriegens with “HotScriptase RT”.
Fluorescence over cycle number is shown AGA is represented by the pink curve, AGG by the dark blue one, CGG
is colored in light blue, GAA in green. The orange curve shows TCC and red TGC. The black curve shows the
negative control. The experiment was carried out in duplicates.

As in the experiment before, codon AGA showed the highest intensity and the lowest Ct value
(17.08). GAA, which normally should be the tRNA with the highest concentration, showed a Ct value
of 25.63 followed by TGC with 25.79 and AGG with 25.92. The Ct value of TCC was 28.2. CGG showed
the highest Ct value of 28.98.

Figure 9: The melting curve of the amplification product of the AGA tRNA in V. natriegens is shown. The
mainly generated product has a melting temperature of about 79 ° C. A second amplification product was
detected with a melting temperature of about 88 °C.

Figure 10: The melting curve of the amplification product of the AGG tRNA in V. natriegens is shown. The
mainly generated product has a melting temperature of about 88 ° C.

Figure 11: The melting curve of the amplification product of the CGG tRNA in V. natriegens is shown. The
amplification product has a melting temperature of about 88 ° C.

Figure 12: The melting curve of the amplification product of the GAA tRNA in V. natriegens is shown. The
amplification product has a melting temperature of about 87 ° C.

Figure 13: The melting curve of the amplification product of the TCC tRNA in V. natriegens is shown. The
amplification product has a melting temperature of about 87 ° C.

Figure 14: The melting curve of the amplification product of the TGC tRNA in V. natriegens is shown. The
amplification product has a melting temperature of about 87 ° C.

Figure 9 – 14 show the melting curves of the qPCR products generated using “HotScriptase
RT”. A single peak that there is only one amplified product for each codon. Figure 10 to 14 indicate,
that unspecific by-products may have been amplified.

**tRNA quantification of rare codons in *V. natriegens* with diluted tRNA**

Because the previous experiments did not reveal the expected result, we tried to proof that the method in
itself is able to detect different tRNA concentrations. Therefore the purified RNA ([Purification\_of\_RNA\_smaller\_than\_200\_nucleotides.pdf](https://static.igem.org/mediawiki/2019/3/3d/T--Stuttgart--Purification_of_RNA_smaller_than_200_nucleotides.pdf)) was diluted 1:1, 1:10 and 1:100 and prepared as usual (<Deacetylation_and_precipitation_of_tRNA.pdf>, <Hybridization_and_ligation_of_tRNAs.pdf>,
[Reverse\_transcription\_of\_the\_specific\_tRNA\_species.pdf](https://static.igem.org/mediawiki/2019/9/9f/T--Stuttgart--Reverse_transcription_of_the_specific_tRNA_species.pdf), [RT\_qPCR.pdf](https://static.igem.org/mediawiki/2019/7/73/T--Stuttgart--RT_qPCR.pdf)). If sample A
showed a Ct of 8 and sample B showed a Ct of 11, sample A contained 23 = 8
times more template. Therefore, for a 10-fold dilution a difference in Ct values of around √10
was expected.

Figure 15: Quantification of diluted tRNAs of the two codons AGA und AGG in *V. natriegens* with
RT-qPCR: Fluorescence over cycle number is shown. AGA 1:1 diluted is represented by pink color. AGA 1:10
diluted by dark blue and 1:100 diluted by light blue. GAA 1:1 is represented by the orange curve, GAA 1:10
by red and GAA 1:100 by dark grey. Also, negative controls are shown: AGA in green and GAA in black.
The experiment was carried out in triplicates. Average and standard deviation are shown.

Figure 15 shows, that our assumption was fulfilled. The Ct values for AGA increased from 10.94 of
the undiluted concentration over 13.17 (1:10) to the 1:100 dilution with a Ct value of 16.56. The
results for GAA are quite similar: lowest Ct value was 24.15 (1:1) and increased to 27.35 (1:10)
and 31.29 (1:100).

After we were able to distinguish tRNA concentrations with a dilution factor of 1:10 significantly, we tried
to distinguish dilutions with factor 1:2, 1:4, 1:8 an 1:16.  Because it was crucial for the results
that each component of the qPCR was pipetted in the right amount, all ingredients were pipetted by a
dispensing robot (iDOT, Dispendix GmbH, Stuttgart, Germany).

Figure 16: Quantification of diluted tRNAs of the AGA in *V. natriegens* with RT-qPCR: Fluorescence
over cycle number is shown. AGA 1:1 dilution: pink, AGA 1:2 dilution: dark blue, AGA 1:4 dilution: light
blue, AGA 1:8 dilution: green, AGA 1:16 dilution: orange, AGA negative control: red. Average of three
technical triplicates and standard deviation are shown.

Figure 17: quantification of diluted tRNAs of the AGG in *V. natriegens* with RT-qPCR: Fluorescence
over cycle number is shown. AGG 1:1 dilution: pink, AGG 1:2 dilution: dark blue, AGG 1:4 dilution: light
blue, AGG 1:8 dilution: green, AGG 1:16 dilution: orange, AGG negative control: red. Average of three
technical triplicates and standard deviation are shown.

Figure 18: quantification of diluted tRNAs of the CGG  in *V. natriegens* with RT-qPCR:
Fluorescence over cycle number is shown. CGG 1:1 dilution: pink, CGG 1:2 dilution: dark blue, CGG 1:4
dilution: light blue, CGG 1:8 dilution: green, CGG 1:16 dilution: orange, CGG negative control: red. Average
of three technical triplicates and standard deviation are shown.

Figure 19: quantification of diluted tRNAs of the GAA in *V. natriegens* with RT-qPCR: Fluorescence
over cycle number is shown. GAA 1:1 dilution: pink, GAA 1:2 dilution: dark blue, GAA 1:4 dilution: light
blue, GAA 1:8 dilution: green, GAA 1:16 dilution: orange, GAA negative control: red. Average of three
technical triplicates and standard deviation are shown.

Figure 20: quantification of diluted tRNAs of the TCC  in *V. natriegens* with RT-qPCR:
Fluorescence over cycle number is shown. TCC 1:1 dilution: pink, TCC 1:2 dilution: dark blue, TCC 1:4
dilution: light blue, TCC 1:8 dilution: green, TCC 1:16 dilution: orange, TCC negative control: red. Average
of three technical triplicates and standard deviation are shown.

Figure 21: quantification of diluted tRNAs of the TGC  in *V. natriegens* with RT-qPCR:
Fluorescence over cycle number is shown. TGC 1:1 dilution: pink, TGC 1:2 dilution: dark blue, TGC 1:4
dilution: light blue, TGC 1:8 dilution: green, TGC 1:16 dilution: orange, TGC negative control: red. Average
of three technical triplicates and standard deviation are shown.

Figures 16 – 21 show the results of the second dilution experiment. Comparing Ct values of
each tRNA species show significant differences between the original samples and the 1:2 dilution for the
rare codons CGG (fig. 18), GAA (fig. 19) and TGC (fig. 21). For the rare codons TCC (fig. 20) and AGG (fig.
17) significant differences in the Ct values between the original sample and the 1:4 dilution
could be shown. A significant difference for the rare codon AGA (fig. 16) could be determined between the
original sample and 1:8 dilution.

Significance was calculated by one way ANOVA followed by a posthoc analysis.

**Comparison of tRNA concentration of rare codons in different *E. coli* strains**

Actually, the goal was to compare the tRNA concentration of *V. natriegens* wildtype with the tRNA
concentration of our codon-optimized strain. The optimized strain was supposed to produce higher amounts of
rare tRNA species. Because the cloning turned out quite difficult, we decided to apply the qPCR method to
other strains. Therefore, *E. coli* BL21DE3, *E. coli* Bl21DE3 pRARE 2 and
*E. coli* Rosetta were cultivated ([Protocol\_Cultivation\_Escherichia\_coli\_for\_RNA\_purification.pdf](https://static.igem.org/mediawiki/2019/6/64/T--Stuttgart--Protocol_Cultivation_Escherichia_coli_for_RNA_purification.pdf)). The strain carrying the pRARE 2 plasmid, should produce higher amounts of rare tRNA species. In case of
the Rosetta strain, the pRARE cassette was integrated into the genome. RNA was purified ([Purification\_of\_RNA\_smaller\_than\_200\_nucleotides.pdf](https://static.igem.org/mediawiki/2019/3/3d/T--Stuttgart--Purification_of_RNA_smaller_than_200_nucleotides.pdf)) and treated as in the experiments with *V. natriegens* (<Deacetylation_and_precipitation_of_tRNA.pdf>, <Hybridization_and_ligation_of_tRNAs.pdf>,
[RT\_qPCR\_without\_prior\_execution\_of\_a\_reverse\_transcription.pdf](https://static.igem.org/mediawiki/2019/8/85/T--Stuttgart--RT_qPCR_without_prior_execution_of_a_reverse_transcription.pdf)). In this experiment, tRNAs of the following rare codons were tested: AGA, AGG, CCC and CUA.

The results are shown in figures 21 – 28 Ct values from *E. coli* pRARE 2 and
Rosetta are quite similar, but the values of the wildtype are always lower, which means that the
concentration is higher. This result does not comply with the assumption, that *E. coli* pRARE 2 and
Rosetta contain higher amounts of these tRNA species.

Figure 21: Comparison of AGA tRNA amplification behavior of *E. coli* BL21DE3 (pink),
*E. coli* Bl21DE3 pRARE 2 (dark blue) and *E. coli* Rosetta (light blue). The negative
control is shown in black. Measurements were carried out in technical triplicates. Averages and standard
deviations are shown.

Figure 22: Melting curve of AGA tRNA amplification mix quantity of *E. coli* BL21DE3 (green),
*E. coli* Bl21DE3 pRARE 2 (pink) and *E. coli* Rosetta (dark blue). The amplification
product has a melting temperature of about 86 °C. Averages and standard deviations of three technical
triplicates are shown.

Figure 23: Comparison of AGG tRNA amplification behavior of *E. coli* BL21DE3 (pink),
*E. coli* Bl21DE3 pRARE 2 (dark blue) and *E. coli* Rosetta (light blue). The negative
control is shown in black. Measurements were carried out in technical triplicates. Averages and standard
deviations are shown.

Figure 24: Melting curve of AGG tRNA amplification mix quantity of *E. coli* BL21DE3 (green),
*E. coli* Bl21DE3 pRARE 2 (pink) and *E. coli* Rosetta (dark blue). The amplification
product has a melting temperature of about 84,5 °C. Averages and standard deviations of three technical
triplicates are shown.

Figure 25: Comparison of CCC tRNA amplification behavior of *E. coli* BL21DE3 (pink),
*E. coli* Bl21DE3 pRARE 2 (dark blue) and *E. coli* Rosetta (light blue). The negative
control is shown in black. Measurements were carried out in technical triplicates. Averages and standard
deviations are shown.

Figure 26: Melting curve of CCC tRNA amplification mix quantity of *E. coli* BL21DE3 (green),
*E. coli* Bl21DE3 pRARE 2 (pink) and *E. coli* Rosetta (dark blue). The amplification
product has a melting temperature of about 86 °C. Averages and standard deviations of three technical
triplicates are shown.

Figure 27: Comparison of CUA tRNA amplification behavior of *E. coli* BL21DE3 (pink),
*E. coli* Bl21DE3 pRARE 2 (dark blue) and *E. coli* Rosetta (light blue). The negative
control is shown in black. Measurements were carried out in technical triplicates. Averages and standard
deviations are shown.

Figure 28: Melting curve of CCC tRNA amplification mix quantity of *E. coli* BL21DE3 (green),
*E. coli* Bl21DE3 pRARE 2 (pink) and *E. coli* Rosetta (dark blue). The amplification
product has a melting temperature of about 86 °C. Averages and standard deviations of three technical
triplicates are shown.

## Outlook

The method shows promising results regarding detection of different tRNA concentration up to the range of
very small concentration differences in diluted samples (fig. 15 – 21), nevertheless the method still
needs to be further optimized. To make sure, the right amplification product for each codon is generated, it
would be necessary to apply next generation sequencing. The difficulty that one faces here is the short
length of the amplificate. Also, a reference method would be needed to confirm our results. As there is no
method for specific tRNA quantification, another option would be, to measure artificially synthesized tRNA
mixtures of known concentrations. Alternatively, deep sequencing could be used to bioinformatically examine
the entire RNA for tRNAs contained in this RNA.

## Growth curves *Vibrio natriegens*

## Results

Vibrio natriegens was cultivated and samples were taken as described ([Protocol\_Growth\_Curve\_Vibrio\_natriegens.pdf](https://static.igem.org/mediawiki/2019/f/f0/T--Stuttgart--Protocol_Growth_Curve_Vibrio_natriegens.pdf)). The results are shown in figure 1. *V. natriegens* grew with a maximum growth rate of
µmax= 2,45 h-1 and reached a doubling time of
td = 16,99 minutes.

Figure 1
: Growth curve *V. Natriegens*. The growth curve for the first biological replicate is in pink,
the second biological replicate is shown with the dark blue curves and the thrid one in a light blue
colour.
Experiment was carried out in biological triplicates, the error bars represent the standard deviation.

## Cloning of tRNA fragments into pSB1C3

The tRNA fragments were synthesized by IDT and amplified by PCR according to the PCR protocol ([Protocol\_PCR.pdf](https://static.igem.org/mediawiki/2019/f/f4/T--Stuttgart--Protocol_PCR.pdf)). The amplified tRNA fragments were validated via agarose gel electrophoresis ([Stuttgart--Protocol\_Agarose\_Gel.pdf](https://static.igem.org/mediawiki/2019/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf)). Looking at Figure 1 all tRNA fragments showed a clear band at the desired height.

![](https://static.igem.org/mediawiki/2019/thumb/8/86/T--Stuttgart--Amplified_tRNA_fragments.png/592px-T--Stuttgart--Amplified_tRNA_fragments.png)

Figure 1: Amplified tRNA fragments. The tRNA fragments AGA, CGC, TGC, TCC and AGG were amplified via
PCR. The PCR products were separated by agarose gel electrophoresis. A 2% agarose gel was prepared and
10 µL were loaded for each probe ((1): AGA, (2): CGC, (3): TGC, (4): TCC, (5): AGG). 3 µL of
Hyberladder 1 kb Bioline were loaded as a marker (M). The gel was run at 90 V for 1 hour and stained
using MidoriGreen.

In a first step, the tRNA fragments were cloned into the pSB1C3 vector. The pSB1C3 and the tRNA
fragments AGA, AGG, CGG, TGC, TCC and a combined tRNA fragment containing all 5 tRNAs were digested
using the restriction enzymes XbaI and SpeI.
After purification, ([Protocol\_Clean\_and\_Concentrate.pdf](https://static.igem.org/mediawiki/2019/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf)) the digested fragments were ligated using the T4 DNA ligase (see
[Protocol\_BioBrick\_Cloning.pdf](https://static.igem.org/mediawiki/2019/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf)). The ligated DNA fragments were transformed into DH5α ([Protocol\_Transformation.pdf](https://static.igem.org/mediawiki/2019/8/83/T--Stuttgart--Protocol_Transformation.pdf)). The plasmid obtained from the colonies ([Protocol\_Plasmid\_Preparation.pdf](https://static.igem.org/mediawiki/2019/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf)) was digested with XbaI to gain linear Plasmid and separated by agarose gel electrophoresis ([Protocol\_Agarose\_Gel.pdf](https://static.igem.org/mediawiki/2019/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf)). Looking at Figure 2 the obtained plasmids showed no insert, only pSB1C3. Also visible is, that
despite the digestion, circular, supercoiled and open circular structures of the plasmid are still
present. This indicates inefficient digestion by the restriction enzymes.

![](https://static.igem.org/mediawiki/2019/thumb/8/8f/T--Stuttgart--Cloning_of_tRNA_fragments_into_pSB1C3.png/722px-T--Stuttgart--Cloning_of_tRNA_fragments_into_pSB1C3.png)

Figure 2: Cloning of tRNA fragments into pSB1C3. The pSB1C3 and the tRNA fragments were digested using
the restriction enzymes XbaI and SpeI. After purification, the digested fragments were ligated using the
T4 DNA ligase. The ligated DNA fragments were transformed into DH5α and subsequently prepared. The
obtained plasmids were digested with XbaI before the agarose gel electrophoresis. A 1% agarose gel was
prepared and 10 µL were loaded for each probe ((1): AGA, (2): AGG, (3): CGG, (4): TGC, (5): TCC,
(6): Combined tRNA fragment). As a control (C) the linear pSB1C3 was loaded. 3 µL of GeneRuler,
1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using
GelRed.

Since the cloning showed inefficient digestion by the enzymes XbaI and SpeI, it was performed again with
the enzymes EcoRI-HF and PstI. The pSB1C3 and the tRNA fragments AGA, AGG, CGG, TGC, TCC and a combined
tRNA fragment were digested using the restriction enzymes EcoRI-HF and PstI.
After purification, ([Protocol\_Clean\_and\_Concentrate.pdf](https://static.igem.org/mediawiki/2019/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf)) the digested fragments were ligated using the T4 DNA ligase (see
[Protocol\_BioBrick\_Cloning.pdf](https://static.igem.org/mediawiki/2019/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf)). The ligated DNA fragments were transformed into DH5α cells ([Protocol\_Transformation.pdf](https://static.igem.org/mediawiki/2019/8/83/T--Stuttgart--Protocol_Transformation.pdf)). The plasmid obtained from the colonies ([Protocol\_Plasmid\_Preparation.pdf](https://static.igem.org/mediawiki/2019/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf)) was digested with EcoRI-HF to gain linear Plasmid and separated by agarose gel electrophoresis ([Protocol\_Agarose\_Gel.pdf](https://static.igem.org/mediawiki/2019/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf)). The agarose gel revealed no successful cloning as all obtained plasmids showed no insert, only
pSB1C3 (data not shown).

## Cloning of tRNA fragments into ptRNA\_backbone via BioBrick Cloning

In a first step, the self-designed linear ptRNA\_backbone from IDT was ligated according to the NEB
Ligation Protocol with T4 DNA Ligase (see
[T--Stuttgart--Blunt\_End\_Ligation.pdf](https://static.igem.org/mediawiki/2019/2/2e/T--Stuttgart--Blunt_End_Ligation.pdf)). Afterward, the ligated ptRNA-backbone was transformed in *E. coli* DH5a cells (see
[Protocol\_Transformation.pdf](https://static.igem.org/mediawiki/2019/8/83/T--Stuttgart--Protocol_Transformation.pdf)). Successfully transformed DH5α cells were selected on LB agar plates containing tetracycline.
The next day the circular ptRNA\_backbone was prepared from the colonies according to the Plasmid
Preparation protocol ([Protocol\_Plasmid\_Preparation.pdf](https://static.igem.org/mediawiki/2019/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf)). The plasmid obtained from the colonies was digested with EcoRI-HF to gain linear Plasmid and
separated by agarose gel electrophoresis ([Protocol\_Agarose\_Gel.pdf](https://static.igem.org/mediawiki/2019/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf)). Looking at Figure 3 all plasmids run at the desired length which corresponds to the length of the
ptRNA\_backbone 2159 bp.

![](https://static.igem.org/mediawiki/2019/thumb/9/9f/T--Stuttgart--Cloning_of_ptRNA_backbone.png/510px-T--Stuttgart--Cloning_of_ptRNA_backbone.png)

Figure 3: Cloning of ptRNA\_backbone. The linear ptRNA\_backbone fragment from IDT was ligated using the
T4 DNA Ligase. The ligated ptRNA-backbone was transformed into DH5α and subsequently prepared. The
obtained plasmids were digested with EcoRI-HF before the agarose gel electrophoresis. A 1% agarose gel
was prepared and 10 µL were loaded for each probe ((1): ptRNA\_backbone gBlock from IDT, (2):
colony 2, (3): colony 3, (4): colony 4, (5): colony 5, (6): colony 6). 3 µL of GeneRuler, 1kb Plus
DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using GelRed.

The ptRNA\_backbone and the previously amplified tRNA fragments AGA, AGG, CGG, TGC, TCC and a combined
tRNA fragment were digested using the restriction enzymes EcoRI-HF and PstI. After purification, ([Protocol\_Clean\_and\_Concentrate.pdf](https://static.igem.org/mediawiki/2019/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf)) the digested fragments were ligated using the T4 DNA ligase (see
[Protocol\_BioBrick\_Cloning.pdf](https://static.igem.org/mediawiki/2019/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf)). The ligated DNA fragments were transformed into DH5α ([Protocol\_Transformation.pdf](https://static.igem.org/mediawiki/2019/8/83/T--Stuttgart--Protocol_Transformation.pdf)). The plasmid obtained from the colonies ([Protocol\_Plasmid\_Preparation.pdf](https://static.igem.org/mediawiki/2019/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf)) was digested with EcoRI-HF to gain linear Plasmid and separated by agarose gel electrophoresis ([Protocol\_Agarose\_Gel.pdf](https://static.igem.org/mediawiki/2019/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf)). Figure 4 reveals no successful cloning as all obtained Plasmids show no insert, only ptRNA\_backbone.

![](https://static.igem.org/mediawiki/2019/thumb/6/6a/T--Stuttgart--Cloning_of_tRNA_fragments_into_ptRNA_backbone.png/576px-T--Stuttgart--Cloning_of_tRNA_fragments_into_ptRNA_backbone.png)

Figure 4: Cloning of tRNA fragments into ptRNA\_backbone. The ptRNA\_backbone and the tRNA fragments were
digested using the restriction enzymes EcoRI-HF and PstI. After purification, the digested fragments
were ligated using the T4 DNA ligase. The ligated DNA fragments were transformed into DH5α and
subsequently prepared. The obtained plasmids were digested with EcoRI-HF before the agarose gel
electrophoresis. A 1% agarose gel was prepared and 10 µL were loaded for each probe ((1): AGA,
(2): AGG, (3): CGG, (4): TGC, (5): TCC, (6): Combined tRNA fragment). As a control (C) the linear
ptRNA\_backbone was loaded. 3 µL of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The
gel was run at 90 V for 1 hour and stained using GelRed.

This BioBrick cloning was repeated several times using different EcoRI-HF and PstI stocks. Following
transformation in DH5α revealed no successful cloning. The colonies obtained showed no insert in
an agarose gel and only ptRNA\_backbone. Following transformation in competent Vibrio natriegens cells
also revealed no successful cloning (data not shown).

## Cloning of tRNA fragments into ptRNA\_backbone via Gibson Assembly

Cloning of tRNA fragments into ptRNA\_backbone was also performed using Gibson Assembly. Gibson Assembly
was conducted according to the protocol Gibson Assembly ([Protocol\_Gibson\_Assembly.pdf](https://static.igem.org/mediawiki/2019/6/6c/T--Stuttgart--Protocol_Gibson_Assembly.pdf)). The Gibson reaction was transformed into competent DH5α cells ([Protocol\_Transformation.pdf](https://static.igem.org/mediawiki/2019/8/83/T--Stuttgart--Protocol_Transformation.pdf)). The plasmid obtained from the colonies ([Protocol\_Plasmid\_Preparation.pdf](https://static.igem.org/mediawiki/2019/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf)) was digested with EcoRI-HF to gain linear Plasmid and separated by agarose gel electrophoresis ([Protocol\_Agarose\_Gel.pdf](https://static.igem.org/mediawiki/2019/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf)). Figure 5 reveals no successful cloning as all obtained Plasmids show no insert, only ptRNA\_backbone.

![](https://static.igem.org/mediawiki/2019/thumb/8/82/T--Stuttgart--Gibson_Assembly_of_tRNA_fragments_into_ptRNA_backbone.png/527px-T--Stuttgart--Gibson_Assembly_of_tRNA_fragments_into_ptRNA_backbone.png)
Figure 5: Gibson Assembly of tRNA fragments into ptRNA\_backbone. Gibson Assembly was performed
according to the Gibson Assembly Protocol. The Gibson Assembly reaction was transformed into DH5α
and subsequently prepared. The obtained plasmids were digested with EcoRI-HF before the agarose gel
electrophoresis. A 1 % agarose gel was prepared and 10 µL were loaded for each probe ((1): AGA,
(2): AGG, (3): CGG, (4): TGC, (5): TCC). As a control (C) the linear ptRNA\_backbone was loaded. 3
µL of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1
hour and stained using GelRed.

Cloning of tRNA fragments into ptRNA\_backbone via Gibson Assembly was repeated with another Gibson
Assembly Master Mix and revealed no successful cloning. The colonies obtained showed no insert in an
agarose gel and only ptRNA\_backbone (data not shown).

## Isolation of *Vibrio natriegens* DSM 759 genome chr.1 and amplification of tRNAs

As an alternative to cloning of tRNA fragments provided by IDT, the tRNAs AGA, AGG, CGG, TGC and TCC
were amplified from the
*Vibrio natriegens* DSM 759 genome. Therefore, the *Vibrio natriegens* DSM 759 genome
chr.1 was isolated according to the gDNA Extraction protocol ([gDNA\_extraction.pdf](https://static.igem.org/mediawiki/2019/c/c0/T--Stuttgart--gDNA_extraction.pdf)). The tRNA fragments AGA, AGG, CGG, TGC, TCC were amplified from the *Vibrio natriegens* DSM
759 genome via PCR with appropriate primers (see
 [Protocol\_PCR.pdf](https://static.igem.org/mediawiki/2019/f/f4/T--Stuttgart--Protocol_PCR.pdf)).
Looking at Figure 6 all tRNA fragments showed a clear band at the desired height. The tRNA fragments
were extracted from the agarose gel according to the gel extraction protocol ([Protocol\_Gel\_Extraction.pdf](https://static.igem.org/mediawiki/2019/3/32/T--Stuttgart--Protocol_Gel_Extraction.pdf)).

![](https://static.igem.org/mediawiki/2019/thumb/3/31/T--Stuttgart--Amplification_of_tRNAs_from_Vibrio_natriegens_genome.png/800px-T--Stuttgart--Amplification_of_tRNAs_from_Vibrio_natriegens_genome.png)
Figure 6: Amplification of tRNAs from the Vibrio natriegens DSM 759 genome. The Vibrio natriegens DSM
759 genome chr.1 was isolated according to the Protocol gDNA Extraction. The tRNA fragments AGA, AGG,
CGG, TGC, TCC were amplified from the Vibrio natriegens DSM 759 genome via PCR with appropriate primers.
A 1.5 % agarose gel was prepared and 10 µL were loaded for each probe ((1): AGA, (2): AGG, (3):
CGG, (4): TGC, (5): TCC). 3 µL of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The
gel was run at 90 V for 1 hour and stained using GelRed.

## Cloning of tRNA fragments (amplified from *Vibrio natriegens* DSM 759 genome) into ptRNA\_backbone via BioBrick Cloning

The ptRNA\_backbone and the previously amplified tRNA fragments AGA, AGG, CGG, TGC, TCC from the
*Vibrio natriegens* DSM 759 genome were digested using the restriction enzymes EcoRI-HF and PstI.
After purification, ([Protocol\_Clean\_and\_Concentrate.pdf](https://static.igem.org/mediawiki/2019/3/3f/T--Stuttgart--Protocol_Clean_and_Concentrate.pdf)) the digested fragments were ligated using the T4 DNA ligase (see
[Protocol\_BioBrick\_Cloning.pdf](https://static.igem.org/mediawiki/2019/0/03/T--Stuttgart--Protocol_BioBrick_Cloning.pdf)). The ligated DNA fragments were transformed into competent DH5α cells ([Protocol\_Transformation.pdf](https://static.igem.org/mediawiki/2019/8/83/T--Stuttgart--Protocol_Transformation.pdf)). The plasmid obtained from the colonies ([Protocol\_Plasmid\_Preparation.pdf](https://static.igem.org/mediawiki/2019/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf)) was digested with EcoRI-HF and PstI to release inserted tRNA fragments from the ptRNA\_backbone and
gain linear plasmid. The DNA fragments were separated by agarose gel electrophoresis ([Protocol\_Agarose\_Gel.pdf](https://static.igem.org/mediawiki/2019/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf)). Looking at Figure 7 no insert band was visible for the obtained plasmids, only ptRNA\_backbone,
suggesting no successful cloning.

![](https://static.igem.org/mediawiki/2019/thumb/5/5e/T--Stuttgart--Biobrick_cloning_of_tRNA_fragments_into_ptRNA_backbone.png/521px-T--Stuttgart--Biobrick_cloning_of_tRNA_fragments_into_ptRNA_backbone.png)

Figure 7: BioBrick cloning of tRNA fragments into ptRNA\_backbone. The tRNA fragments were previously
amplified from the Vibrio natriegens DSM 759 genome. The ptRNA\_backbone and the tRNA fragments were
digested using the restriction enzymes EcoRI-HF and PstI. After purification, the digested fragments
were ligated using the T4 DNA ligase. The ligated DNA fragments were transformed into DH5α and
subsequently prepared. The obtained plasmids were digested with EcoRI-HF and PstI before the agarose gel
electrophoresis. A 1% agarose gel was prepared and 10 µL were loaded for each probe ((1): AGA,
(2): AGG, (3): CGG, (4): TGC, (5): ACC). As a control (C) the linear ptRNA\_backbone was loaded. 3
µL of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1
hour and stained using GelRed.

Following transformation in competent Vibrio natriegens cells also revealed no successful cloning (data
not shown).

### Cloning of tRNA fragments (amplified from *Vibrio natriegens* DSM 759 genome) into ptRNA\_backbone via NEBuilder HiFi DNA Assembly

The tRNA fragments were previously amplified from
*Vibrio natriegens* DSM 759 genome. The NEBuilder HiFi DNA Assembly was performed according to
the Protocol NEBuilder HiFi DNA Assembly (<https://static.igem.org/mediawiki/2019/2/25/T--Stuttgart--Protocols_NEBuilder_HiFi_DNA_Assembly.pdf>). Following transformation into competent DH5α cells revealed no successful cloning as no
colonies were obtained. Following transformation in competent *Vibrio natriegens* cells also
revealed no successful cloning (data not shown).

### Cloning of tRNA fragments (amplified from *Vibrio natriegens* DSM 759 genome) into ptRNA\_backbone via Gibson Assembly

Gibson Assembly was performed according to the protocol Gibson Assembly (<https://static.igem.org/mediawiki/2019/6/6c/T--Stuttgart--Protocol_Gibson_Assembly.pdf>). Due to the orientation in the *Vibrio natriegens* DSM 759 genome only the tRNA fragments AGG
and TGC could be used for Gibson Assembly. The Gibson reaction was transformed into competent DH5α
cells ([Protocol\_Transformation.pdf](https://static.igem.org/mediawiki/2019/8/83/T--Stuttgart--Protocol_Transformation.pdf)). The plasmid obtained from the colonies ([Protocol\_Plasmid\_Preparation.pdf](https://static.igem.org/mediawiki/2019/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf)) was digested with EcoRI-HF and PstI to release inserted tRNA fragments from the ptRNA\_backbone and
gain linear plasmid. The DNA fragments were separated by agarose gel electrophoresis ([Protocol\_Agarose\_Gel.pdf](https://static.igem.org/mediawiki/2019/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf)). Looking at Figure 8 no insert band was visible for the obtained plasmids, only ptRNA\_backbone,
suggesting no successful cloning.

![](https://static.igem.org/mediawiki/2019/thumb/9/99/T--Stuttgart--Gibson_Assembly_of_tRNA_fragments_-2.png/512px-T--Stuttgart--Gibson_Assembly_of_tRNA_fragments_-2.png)

Figure 8: Gibson Assembly of tRNA fragments into ptRNA\_backbone. Gibson Assembly was performed according
to the Gibson Assembly Protocol. The tRNA fragments were previously amplified from the Vibrio natriegens
DSM 759 genome. The Gibson Assembly reaction was transformed into DH5α and subsequently prepared.
The obtained plasmids were digested with EcoRI-HF and PstI before the agarose gel electrophoresis. A 1%
agarose gel was prepared and 10 µL were loaded for each probe ((1): AGG colony 1, (2): AGG colony
2, (3): TGC colony 1, (4): TGC colony 2). 3 µL of GeneRuler, 1kb Plus DNA Ladder was loaded as a
marker (M). The gel was run at 90 V for 1 hour and stained using GelRed.

Cloning of tRNA fragments into ptRNA\_backbone via Gibson Assembly was repeated with another Gibson
Assembly Master Mix and revealed no successful cloning. The colonies obtained showed no insert in an
agarose gel and only ptRNA\_backbone. Following transformation of the Gibson reaction in competent
*Vibrio natriegens* cells also revealed no successful cloning (data not shown).

### Outlook

Regarding all experiments, several different cloning methods did not result in successful insertion of
tRNA fragments in ptRNA\_backbone or pSB1C3. Therefore, we commissioned Synbio-Technologies to perform
the cloning. After three weeks the company was also unable to perform the cloning successfully. Since
the company has newly synthesized the tRNA inserts and the ptRNA\_backbone there should be no error in
the DNA sequences and cloning. One explanation for unsuccessful cloning could be the toxicity of the
tRNA constructs. Only cells that have taken up the ptRNA\_backbone without insert survive and result in
colonies due to spurious transcription. Impaired cell function can be due to de-repression of spurious
transcription in bacteria. 1 Constitutive transcription initiation can lead to titration of
RNA polymerase and global downregulation in host gene expression. 2

For future experiments one conceivable approach would be the genomic integration of tRNA genes in order
to increase the tRNA concentration without a plasmid system.

### References

1. Wade JT, Grainger DC. Spurious transcription and its impact on cell function.
   *Transcription*. 2018. doi:10.1080/21541264.2017.1381794
2. Lamberte LE, Baniulyte G, Singh SS, et al. Horizontally acquired AT-rich genes in Escherichia coli
   cause toxicity by sequestering RNA polymerase. *Nat Microbiol*. 2017.
   doi:10.1038/nmicrobiol.2016.249

# Transformation of pRARE into *Vibrio natriegens*

As an alternative to cloning our self-designed ptRNA\_backbone containing tRNAs of rare codons into
*Vibrio natriegens* we used the pRARE plasmid. It carries genes for several rare tRNAs in
*Escherichia coli*, which allows their concentration to be increased. The pRARE plasmid was prepared
from *E. coli* Rosetta and transformed into *Vibrio natriegens (*[Protocol\_Transformation.pdf](https://static.igem.org/mediawiki/2019/8/83/T--Stuttgart--Protocol_Transformation.pdf)). Growth curves of the wild type *Vibrio natriegens* and *Vibrio natriegens* containing
pRARE were performed ([Protocol\_Growth\_Curve\_Vibrio\_natriegens.pdf](https://static.igem.org/mediawiki/2019/f/f0/T--Stuttgart--Protocol_Growth_Curve_Vibrio_natriegens.pdf)). The growth curves represented in Figure 1 show no differences in the growth velocity between both
*Vibrio natriegens* strain variants. Thus, the enhanced expression of tRNAs has no obvious negative
effect on the metabolic activity of *Vibrio natriegens* cells.

Figure 1:  Growth curve of Vibrio natriegens containing pRARE. The pRARE plasmid was prepared from E.
coli Rosetta and transformed into Vibrio natriegens. Two colonies were selected for each Vibrio natriegens
strain variants and a 5 mL starter culture of BHIN was inoculated. The culture was grown at 37 °C and
150 rpm overnight. 150 mL BHIN (500 mL shaking flask) were inoculated with preculture to an initial OD600 nm
of 0.05. Vibrio natriegens was cultivated at 37 °C and 150 rpm. OD600nm was measured every 30
minutes in duplicates.

In order to investigate the possible positive effects of increased tRNA expression on protein expression,
special sfGFP variants were supposed to be developed as explained in section Mutagenesis of
sfGFP.

## Mutagenesis of sfGFP

The vector pYTK047 containing sfGFP-Sequence was kindly provided by iGEM Team Marburg. To evaluate the
expression of proteins containing rare codons (AGA, AGG, CGG, TGC, TCC) the tags of rare codons were
introduced into the pYTK047. During the expression, the tags are fused to the N-terminus of the protein
sfGFP. Thereby they determine the initial translation velocity of the expression of the fusion protein.

To insert the tags of rare codons into the pYTK047 genome, PCR with specialized primers was performed (for
more detailed information see
[Q5\_Site-directed\_mutagenesis.pdf](https://static.igem.org/mediawiki/2019/e/ed/T--Stuttgart--Q5_Site-directed_mutagenesis.pdf)). To remove the unamplified template vector the PCR reaction was digested with Dpn1 and separated by
agarose gel electrophoresis ([Protocol\_Agarose\_Gel.pdf](https://static.igem.org/mediawiki/2019/b/ba/T--Stuttgart--Protocol_Agarose_Gel.pdf)). Looking at Figure 1 only the constructs containing the tags AGA (1), AGG (2) and TGC (4) could be
amplified by PCR.

![](https://static.igem.org/mediawiki/2019/4/4e/T--Stuttgart--Amplified_pYTK047_with_tags_of_rare_codons.png)

Figure 1: Amplified pYTK047 with tags of rare codons. pYTK047 with tags of rare codons (AGA, AGG, CGC, TGC
and TCC) were amplified via PCR. The PCR products were separated by agarose gel electrophoresis. A 1 %
agarose gel was prepared and 50 µL were loaded for each probe ((1): pYTK047-AGA, (2): pYTK047-AGG,
(3): pYTK047-CGG, (4): pYTK047-TGC, (5): pYTK047-TCC). 3 µL of Quick-Load® Purple 1 kb Plus DNA
Ladder (NEB) were loaded as a Marker (M). The gel was run at 90 V for 1 hour and stained using GelRed.

The bands of the desired size were cut out of the agarose gel and the DNA was extracted ([Protocol\_Gel\_Extraction.pdf](https://static.igem.org/mediawiki/2019/3/32/T--Stuttgart--Protocol_Gel_Extraction.pdf)). A KLD ligation was performed according to the NEB KLD Enzyme Mix Reaction Protocol to circularize the
extracted linear DNA. After ligation, the pYTK047 with the tags of rare codons was transformed in
*E. coli*  DH5α following the transformation protocol for chemical transformation ([Protocol\_Transformation.pdf](https://static.igem.org/mediawiki/2019/8/83/T--Stuttgart--Protocol_Transformation.pdf)). No colonies were obtained after transformation. The transformation was repeated, but the plasmid was not
transformed successfully. The plasmid was supposed to be isolated from an overnight culture ([Protocol\_Plasmid\_Preparation.pdf](https://static.igem.org/mediawiki/2019/2/2c/T--Stuttgart--Protocol_Plasmid_Preparation.pdf)) and successful introduction of rare codons into plasmid pYTK047 should have been validated through
sequencing. Transformation into *Vibrio natriegens* ([Protocol\_Transformation.pdf](https://static.igem.org/mediawiki/2019/8/83/T--Stuttgart--Protocol_Transformation.pdf)) and following growth curves or expression analysis should reveal if protein expression is impaired when
rare codons are present.

In the last step, we wanted to investigate if the expression of proteins with rare codons can be rescued or
even increased when a higher concentration of tRNAs for those rare codons are present. Therefore, pRARE from
*E. coli* Rosetta was supposed to be transformed into *V. natriegens* together with pYTK047
containing sfGFP with rare codons. The plasmid pRARE codes for the tRNA of proL, leuW, metT, argW, thrT,
glyT tyrU, thrU, argU and ileX and therefore increases their concentrations.

## Media based on algae: first tests determining important substrates

### Medium based on LB

To first determine whether the extract of
*chlorella vulgaris* was a substitute for yeast extract, LB medium and medium containing
*chlorella vulgaris* extract instead of yeast extract were produced.

1. Media were produced ([Protocol\_media\_first\_experiments.pdf](https://2019.igem.org/File%3AT--Stuttgart--Protocol_media_first_experiments.pdf)).
2. Media were inoculated with *escherichia coli* or
   *vibrio natriegens.*
3. After the over night culture, the turbidity of the tubes was observed.

Result: Growth of both bacteria was observed in both media.

### Medium without tryptone

To determine whether *Chlorella vulgaris* extract was able to substitute tryptone as a medium
component, additionally, media containing different concentrations of NaCl
*Chlorella vulgaris* extract and yeast extract were prepared.

1. Media were produced ([Protocol\_media\_first\_experiments.pdf](https://2019.igem.org/File%3AT--Stuttgart--Protocol_media_first_experiments.pdf)).
2. Media were inoculated with *vibrio natriegens.*
3. After the overnight culture, the turbidity of the tubes was observed.

Result: No growth was detected in media without tryptone.

## Autolysis in combination with bead-milling results

### Free amino acid estimation with rFAN assay

Samples from Experiment
[Cell\_extraction\_with\_autolysis\_combined\_with\_bead-milling.pdf](https://static.igem.org/mediawiki/2019/e/ec/T--Stuttgart--Protocol_Cell_extraction_with_autolysis_combined_with_bead-milling.pdf)
were used for the analysis.

Yeast extract is mostly obtained by autolysis 1. In autolysis cells digest their own cell
compounds with their own enzymes 2. The idea was to transfer this commonly used principal on
algae. Therefore, *C. vulgaris* and *C. sorokiniana* were heated to 50 °C in alkaline
or acidic environment for 41 h. To further crack the cell wall, both algae were treated with
bead-milling afterwards. To quantify the success of cell wall disruption free amino acids were measured with
rFAN-assay.

The yield of free amino acids was set into relation with the amount of biomass used in the experiment
(figure 1).

Figure 1: Autolysis and subsequent bead-milling of algae C. vulgaris and C. sorokiniana. The percentage of
free amino acids [%] relates to the biomass used in the experiment.

The highest amounts of free amino acids (4.85 %) were reached with yeast at pH 12. Both algae showed
very low yield in free amino acids. The best results showed *C. sorokiniana* at pH 12. It is
possible, that the amount of glass beads and the size of the glass beads were too little, which led to less
cell wall disruption. Therefore, amino acids would have been retained within the cells. This would explain
the little amounts of free amino acids achieved with this method. Also, *C. vulgaris* and
*C. sorokinia* have a cell wall, in contrast to yeast 3. Cell walls are harder to break,
than a plasma membrane. This could explain the difference between the yeast samples and the algae samples.
Due to the low yield in free amino acids, it was decided to investigate other methods for cell disruption of
algae.

### Anthrone assay to Determine Soluble Carbohydrate Concentration

Similar to the rFAN assay the anthrone assay is a method to detect free monosaccharides in a liquid.
Therefor samples from the experiment

[Experiments\_AnthroneAssay.pdf](https://static.igem.org/mediawiki/2019/f/f3/T--Stuttgart--Experiments_AnthroneAssay.pdf)
were analyzed. Hereby a calibration curve with known amounts of glucose is created (Figure 2, left side).
This calibration curve creates the possibility to calculate the sugar concentration of the samples (Figure
2, right side).

![](https://static.igem.org/mediawiki/2019/thumb/2/2f/T--Stuttgart--FigureAutolysis_in_combination_with_bead-milling_Results2.png/800px-T--Stuttgart--FigureAutolysis_in_combination_with_bead-milling_Results2.png)
Figure 2: Pictures of the anthrone calibration curve as well as the anthrone assay of samples. For the
calibration curve known amounts of glucose is dissolved in water and the optical density at 620 nm is
measured (left side). This can be used to determine the monosaccharide concentration of anthone treated
samples which previously underwent autolysis (pH3 or pH6) with or without subsequent bead-mill treatment
(RKM) (right side).

One can tell from the coloring of the samples in figure 2, that the carbohydrate concentration should differ
very slightly between the samples pH3, pH6, bead mill extraction +pH3 and bead mill extraction +pH6. Due to
the cloudiness of the control sample, a background corrected optical density could not be determined.
Therefore, the coloring scheme served as evaluation for successful carbohydrate determination.

Hereby, bead-mill (RKM) with subsequent autolysis at pH3 was determined to be the method of choice.

### References

1. Kim et al., “Preparation of flavor-enhancing yeast extract using a Saccharomyces cerevisiae strain
   with high RNA content”, Korean J Food Sci Technol, 31 (2) (1999), pp. 475-481.
2. T.L. Babayan, M.G. Bezrukov, “Autolysis in yeasts”, Acta Biotechnol, 5 (2) (1985), pp.
   129-136.
3. van der Rest, M E et al. “The plasma membrane of Saccharomyces cerevisiae: structure, function,
   and biogenesis.” Microbiological reviews vol. 59,2 (1995): 304-22.
4. Takeda, “Classification of Chlorella strains by cell wall sugar composition” Phytochemistry,
   vol. 27, 12, (1988), pp. 3823-3826.
5. [4} Takeda, “Classification of Chlorella strains by cell wall sugar composition”
   Phytochemistry, vol. 27, 12, (1988), pp. 3823-3826.

# Results: Cell wall disruption of *C. vulgaris* and *C. sorokiniana*

For cell wall disruption of *C. vulgaris* and *C. sorokiniana* physical and chemical
approaches were tested. One physical approach was bead milling with different amounts and sizes of glass
beads. Also, one experiment was performed with beads with sharp edges. As an chemical approach for cell wall
disruption, algae were boiled in acid or base at 100 °C for one hour (<https://static.igem.org/mediawiki/2019/e/ec/T--Stuttgart--Protocol_Cell_extraction_with_autolysis_combined_with_bead-milling.pdf>).

To quantify the amount of protein, received from the respective cell wall disruption method, Bradford-Assay
([https://2019.igem.org/File:T--Stuttgart--Experiments\_BradfordAssay.pdf](https://2019.igem.org/File%3AT--Stuttgart--Experiments_BradfordAssay.pdf)) was performed. The amount of protein was related with the amount of used biomass. First we decided to
test physical cell wall disruption (Fig. 1).

![](https://static.igem.org/mediawiki/2019/9/94/T--Stuttgart--Fig1Results_CellWall_Bradford_Anthrone.png)
Figure 1: Percentage of protein [%] related to the used biomass of C. sorokiniana. The cell wall was
disrupted by bead milling with glass beads in size 45-75 µm and 200-300 µm. 50 %
and 80 % of glass beads were tested. The amount of protein was quantified with Bradford-Assay.

The highest amount of protein (15.6 %) was achieved with 80 % of glass beads with a size between
200-300 µm. The smaller glass beads reached up to 14.04 % of protein and were less efficient. A
drastic difference can be seen between 50 % glass beads and 80 % glass beads. With the smaller
beads the amount of glass beads used led to a difference of 11.93 %, similar to the bigger beads with a
difference of 11.77 %. Therefore, 80% of glass beads was used for future cell wall disruption to create
our PhyCoVi medium. Also, the bigger beads led to a higher yield of protein and were therefore chosen to be
the future method for cell wall disruption.

An additional approach of physical cell wall disruption was to use special beads with sharp edges at
different speeds (Fig. 2). The beads had a size of 2 mm.

![](https://static.igem.org/mediawiki/2019/c/cb/T--Stuttgart--Fig2Results_CellWall_Bradford_Anthrone.png)
Figure 2: Cell wall disruption with beads with sharp edges. Percentage of protein [%] is related to the
used biomass of C. sorokiniana and C. vulgaris, respectively. For quantification of Protein, Bradford-Assay
was used.

The highest yield of protein was gained with *C. vulgaris* at speed 8. Speed 4 always led to a lower
yield of protein. *C. sorokiniana* reached a lower yield of protein with 1.19 %. The sharp-edged
beads reached a lower yield of protein compared to the glass beads with a size of 200-300 µm.
Hence, this method was not considered for further use.

To further optimate the cell wall disruption methods and increase the amount of usable sample, cell wall
disruption with acid and base was tested. As bases we used 1 M KOH, 5 M KOH and 1 M NaOH, as
acid 1 M HCl was used (Fig. 3).

![](https://static.igem.org/mediawiki/2019/9/9f/T--Stuttgart--Fig3Results_CellWall_Bradford_Anthrone.png)
Figure 3: Cell wall disruption with 1 M HCl, 1 M NaOH, 1 M KOH and 5 M KOH. The
percentage of protein was related to the used biomass of C. sorokiniana. Samples were boiled for 1 h at
100 °C and later quantified with Bardford-Assay.

The highest yield of protein was reached with 5 M KOH with 9.03%. Lower amounts of protein were gained
with 1M HCl and 1M KOH. NaOH led to the least yield of protein with 3.59 %.

After boiling the solutions had to be neutralized. Therefore, we used 1 M NaOH and 1 M HCl.
Neutralizing HCl with NaOH leads to the formation of NaCl, which is a compound of the common cultivation
medium for *V. natriegens*. Our PhyCoVi medium was primary created to be a medium for
*v. natriegens*. Therefore, we chose cell disruption with 1 M HCl to be the future method.

All samples were also analyzed by the Anthrone Method (<https://static.igem.org/mediawiki/2019/f/f3/T--Stuttgart--Experiments_AnthroneAssay.pdf>), to quantify the amount of free sugars and starch. The anthrone reagent coagulates with sugars and
starch. The more carbohydrates are available, the greener the samples appears and the higher the absorbance
at 620 nm. Concerning different sizes of the glass beads, the small beads with 100 to 200 µm
showed the best results. But also the high speed disruption with sharp edged glass beads around 2.0 mm
showed good results (Fig. 4). Since this option is only available at the Max-Planck-Institute in
Tübingen, we chose the options we had available in Stuttgart.

![](https://static.igem.org/mediawiki/2019/a/a5/T--Stuttgart--Fig4Results_CellWall_Bradford_Anthrone.png)
Figure 4: Amount of Carbohydrates [AU] in disrupted C. vulgaris and C. sorokiniana cells. The cell wall was
disrupted by bead milling with glass beads in size 2.0 mm at high speed, 0.75 to 1.0 mm, 2.0 mm
and 2.1 mm.  The amount of Carbohydrates were quantified with anthrone assay

For the two volume ratios of glass beads to sample, the 80 % of 200 µm glass beads showed
the best result. Generally more and bigger glass beads show better cell disruption.

![](https://static.igem.org/mediawiki/2019/2/2a/T--Stuttgart--Fig5Results_CellWall_Bradford_Anthrone.png)
Figure 5: Amount of Carbohydrates [AU] in disrupted C. sorokiniana cells. The cell wall was disrupted by
bead milling with glass beads in size 45-75 µm and 200-300 µm. 50 % and 80 %
of glass beads were tested. The amount of Carbohydrates were quantified with anthrone assay.

At last the acid and base disruption was examined. As seen in Fig. 6, all bases show low values, with
5 M KOH being unusable. With HCl reaching a similar level like glass beads, we chose this acid as our
method for cell disruption. In combination with glass bead disruption, we hoped to have an efficient method
for cell disruption of micro algaes.

![](https://static.igem.org/mediawiki/2019/7/70/T--Stuttgart--Fig6Results_CellWall_Bradford_Anthrone.png)
Figure 6: Cell wall disruption with 1 M HCl, 1 M NaOH, 1 M KOH and 5 M KOH. Samples
were boiled for 1 h at 100 °C. The amount of Carbohydrates were quantified with anthrone
assay.

## CDW correlation of algae *Chlorella vulgaris* Results

By plotting the measured optical densities against the means of the calculated cellular dry weights, a
correlation was obtained. It is shown in the following figure.

![](https://static.igem.org/mediawiki/2019/a/ad/T--Stuttgart--FigureOD-CDW_correlation_of_algae_Chlorella_vulgaris_Results1.png)
Figure 1: OD-CDW correlation of the algae *Chlorella vulgaris*. Mean of cellular dry weight in g/L
(n=2) was plotted against the measured optical density at 750 nm. Trend line was shown in red.

The trend line in figure 1 is poorly matching the trend of the measurement points. For this reason, the
correlation curve was rejected. For improvement of this experiment, measurements should be performed only by
one experimenter to reduce pipetting errors or other handling mistakes. Also the measurements should be
taken over a longer time period to gain more trust worthy results.

## CDW-OD correlation by dilution Results

In the following table you can see the calculated cell dry weights with the corresponding optical density of
the tubes. Some tubes broke during the experiment so corresponding measurements could not occur.

Table 2: Calculated values of the cellular dry weight in g/l with the corresponding optical density
measured at 750 nm.

| OD | CDW[g/l] |
| --- | --- |
| 4.87 | 0.98 |
| 4.41 | 0.88 |
| 4.44 | 0.91 |
| 4.02 | 0.8 |
| 3.92 | 0.82 |
| 3.52 | 0.68 |
| 3.57 | 0.71 |
| 3.03 | 0.57 |
| 2.65 | 0.49 |
| 2.8 | 0.5 |
| 2.43 | 0.48 |
| 2.56 | 0.46 |
| 2.29 | 0.4 |
| 2.28 | 0.4 |

The cellular dry weight in g/l was then plotted against the optical density measured at 750 nm. The plot
with the corresponding trend line is shown in the following figure.
![](https://static.igem.org/mediawiki/2019/d/df/T--Stuttgart--FigureOD-CDW_correlation_of_Chlorella_vulgaris_by_dilution_Results1.png)
Figure 1: Cellular dry weight in g/l is plotted against the optical density measured at 750 nm. The
linear fit is shown in blue together with its formula.

The slope of the formula further been used for fast estimation of the CDW by measuring the optical
density at 750 nm.

## Results preliminary *Chlorella vulgaris* extraction methods (FACS analysis)

The obtained samples were measured at a flow-cytometer for green fluorescence (intact chlorophyll) as well
as cell size (SSC and FSC). A distinct amount of liquid (50 µL) was measured. Based on the cell size
(intact cells show a clear population) and green fluorescence, viable cells were able to be gated into a
distinct population. With obtaining a distinct number of viable cells within a constant volume we were able
to determine the number of viable cells per µL. The number of cells/µL of the control sample was
set to 100% with the extraction methods (high-pressure-homogenizer passages 1 - 4, mortar, bead-mill,
autoclave and sonification) set in relation of the control. We hereby obtained the percentage of viable
cells for each cell disruption method (Figure 1).

![](https://static.igem.org/mediawiki/2019/2/2b/T--Stuttgart--Bilder_Aufschluss.png)

Figure 1: Measured cell viability of *Chlorella vulgaris* after cell disruption methods. A
distinct amount of cells was used to test each disruption method: high-pressure-homogenizer passage 1 - 4,
mortar, bead-mill, autoclave and sonification. Afterwards 50 µL of the obtained samples were analyzed
at the flow cytometry based on positive green fluorescence signal as well as intact cell signal (FSC, SSC).
The cell count was measured, and the control was set to 100% cell viability with the cell count of the
disruption methods set in relation to the control.

## Conclusion:

In terms of feasibility and up-scaling problems the method we further wanted to analyze was the bead-mill.
With a 50% cell viability it proved to be a rather solid method for cell disruption. We concluded that
further research for the optimal bead size should be conducted as well as a combination with an autolytic
process. The idea was that acid induced autolysis prior to the bead-mill treatment should weaken the strong
microalgae cell wall and therefore decrease cell viability.

## Growth Curves of various bacteria in PhyCoVi Medium

The PhyCoVi medium was tested with various microorganism including
*Vibrio natriegens, Escherichia coli*, *Bacillus subtillis* and
*Corynebacterium glutamicum*. Therefore, optical density was measured at 600 nm with a photometer .
Different medium variations were tested to find the best medium composition. For control
also normal LB medium, Brain-Heart-Infusion (BHIN) medium and medium containing only soy peptone was tested. Medium
composition is shown in table 1.

Table 1: Specification for all media used during the algae based PhyCoVi medium (PCV) validation.

| Ingredients | PCV [g/L] | PCV + Soy Peptone [g/L] | LB medium [g/L] | BHIN [g/L] | Soy Peptone Control [g/L] |
| --- | --- | --- | --- | --- | --- |
| NaCl | 10 or 20 | | | | |
| Algae extract | 30 | 30 | -- | -- | -- |
| Soy Peptone | 10 | 10 | 10 | -- | 10 |
| Yeast extract | -- | -- | 5 | -- | -- |
| BHI | -- | -- | -- | 37 | -- |

To estimate the optimal medium composition for *V. natriegens* different NaCl concentrations were
tested as well as the addition of soy peptone (Figure 1).

![](https://static.igem.org/mediawiki/2019/f/f3/T--Stuttgart--Fig2Demonstrate.png)
Figure 1: Growth curve of *V. natriegens* on PhyCoVi Medium (PCV) and PCV + Soy peptone. LB-Medium,
Brain-Heart infusion Medium (BHIN) and soy peptone served as control. The medium contained different amounts
of NaCl (10 or 20 g/L). Sampling for measuring optical density was performed every 45 minutes over a time course of
315 minutes.

The PCV medium containing additional soy peptone revealed the highest growth of *V. natriegens*. It
even outcompeted the commonly used BHIN medium. *V. natriegens* cultivated in PCV medium without
additional soy peptone showed remarkably less growth. LB and Soy peptone alone showed the least bacterial
growth.

Next, bacterial growth of *Escherichia coli MG1655* on PhyCoVi medium was tested (Figure 2).

![](https://static.igem.org/mediawiki/2019/f/f2/T--Stuttgart--Fig3Demonstrate.png)
Figure 2: Growth curve of *E. coli* in PhyCoVi Medium (PCV) and PCV + Soy peptone. LB-Medium, Brain-Heart
infusion Medium (BHIN) and soy peptone served as control. The medium contained different amounts of NaCl (10
or 20 g/L). Sampling for measuring optical density was performed every 45 minutes over a time course of
315 minutes.

Highest bacterial growth was reached with PCV medium containing soy peptone and 10 g/L NaCl
followed by the 20 g/L NaCl variant. The differences in *E. coli* growth on various media is not as clear
as it was with *V. natriegens* (Figure 1). Also, the adapation phase is longer. Nevertheless, PhyCoVi
medium with soy peptone is applicable for cultivation of *E. coli*.

Further, growth of *Bacillus subtilis* (Figure 3) and
*Corynebacterium glutamicum* (Figure 4) were tested. Only medium containing 10 g/L NaCl was
used.

![](https://static.igem.org/mediawiki/2019/4/48/T--Stuttgart--Fig4Demonstrate.png)
Figure 3: Growth curve of *Bacillus subtillis* on PhyCoVi Medium (PCV) and PCV + soy peptone. LB-Medium,
Brain-Heart infusion Medium (BHIN) and soy peptone served as control. The medium contained 10 g/L NaCl.
Sampling for measurements of the optical density was performed every 45 minutes over a time course of 360 minutes.

*Bacillus subitilis* grew best on BHIN followed by LB medium. PCV medium with soy peptone was still
better than PCV without it. Nevertheless, there was no remarkable increase in absorbance and thus only
little growth of bacteria after 6 h.

![](https://static.igem.org/mediawiki/2019/8/8b/T--Stuttgart--Fig5Demonstrate.png)
Figure 4: Growth curve of *Corynebacterium glutamicum* on PhyCoVi Medium (PCV) and PCV + soy peptone.
LB-Medium, Brain-Heart infusion Medium (BHIN) and soy pepton served as control. The medium contained
10 g/L NaCl. Sampling for measurements of the optical density was performed every 45 minutes over a time course of 360 minutes.

*Corynebacterium glutamicum* grew better on BHIN and LB than on PCV with or without soy peptone. PCV
with additional soy peptone showed higher growth than medium without soy peptone. Comparing
*Bacillus subtilis* with *Corynebacterium glutamicum*, *C. glutamicum* shows better
growth on PCV containing soy peptone. Although both bacteria prefer BHIN and LB medium.

## Sugar HPLC Results

The measurement was performed in six technical replicates of the algae extract batch PCV#2 from
*Chlorella vulgaris*. The molar concentration of glucose, fructose, sucrose and raffinose were
determined. The results of the carbohydrate HPLC measurement are shown in Table 1.

Table 1: Molar concentration [µM] of glucose, fructose, sucrose and raffinose in 30 g/l
*Chlorella vulgaris* extract diluted in dH2O measured in 6 technical replicates.

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| **Carbohydrate** | **PCV#2.1 [µM]** | **PCV#2.2 [µM]** | **PCV#2.3 [µM]** | **PCV#2.4 [µM]** | **PCV#2.5 [µM]** | **PCV#2.6 [µM]** |
| Glucose | 2075.56 | 2040 | 1987.23 | 2199.63 | 2208.8 | 2119.68 |
| Fructose | 707.92 | 681.28 | 654.26 | 639.44 | 625.7 | 593.94 |
| Sucrose | 2.87 | 0 | 0 | 0 | 0 | 0 |
| Raffinose | 0 | 0 | 0 | 0 | 0 | 0 |

We were able to measure glucose and fructose in our samples. Raffinose was not detected at all. Sucrose was
measured only in one sample, so we decided to reject its concentration.

By calculating the averages of the 6 replicates we were able to determine the amount of certain carbohydrate
in gram per 100-gram *Chlorella vulgaris* extract. In 100 g *C. vulgaris* extract we obtained
1.264 g glucose and 0.391 g fructose.

## Amino acid HPLC Results

For this experiment we measured three technical replicates of two batches of our self-made algae extract
from *Chlorella vulgaris* with two concentrations 30 g/L and 60 g/L. The two measurements were
performed to have a detectable amount of amino acids. Histidine in batch PCV#3 was only measured in the 60
g/L sample, he corresponding values were divided by 2 and added to the 30 g/L values. We calculated averages
and standard deviations of the molar concentrations from the HPLC data for all 20 proteinogenic amino acids.
To be able to compare the two batches PCV#2 and PCV#3, we plotted the average concentrations in a bar
diagram. We were not able to distinguish between the peaks of alanine and arginine. For that reason, they
were analyzed as one peak.

![](https://static.igem.org/mediawiki/2019/4/4b/T--Stuttgart--FigureHPLC2.png)

Figure 1: Bar diagram of average molar concentration of 20 proteinogenic amino acids from two batches of
self-made algae extract from *Chlorella vulgaris* diluted in water (30g/l). In blue the prior extract with the
ID: PCV#2. In green the final batch with ID: PCV#3.

It is noteworthy, that the batch PCV#3 shows a brighter spectrum of amino acids, than the prior batch PCV#2.
When harvesting the algae for the batch PCV#3, we tried to optimize the protocol for extraction. The prior
extraction protocol had some issues with product purity. The powder contained some disintegrated algae
contaminants. However, we could achieve an improvement when it comes to amino acid content.

To create a content list for our algae *C.vulgaris* extract, we calculated the mass of amino acids
per 100 gram extract for both batches PCV#2 and PCV#3. The corresponding list is shown in the following
table.

Table 1: List of amino acid content in gram per 100 g *Chlorella vulgaris* extract.

|  |  |  |
| --- | --- | --- |
| **Amino acid** | **g per 100 g PCV#2** | **g per 100 g PCV#3** |
| Asp | 0.070086304 | 0.057490545 |
| Glu | 0.040912751 | 0.049566772 |
| Ser | 0.010175455 | 0.017357337 |
| His | 0 | 0.000597254 |
| Gly | 0.010794219 | 0.022097401 |
| Thr | 0.044205579 | 0.007266782 |
| Tyr | 0 | 0.006698225 |
| Cys | 0 | 0.024702034 |
| Val | 0 | 0.01251605 |
| Met | 0.003853789 | 0.052499327 |
| Trp | 0 | 0.00494474 |
| Phe | 0 | 0.011192791 |
| Ile | 0 | 0.004606983 |
| Leu | 0.00132329 | 0.011335256 |
| Lys | 0 | 0.038419982 |
| Pro | 0.025929325 | 0.018145512 |

The total amount of proteinogenic amino acids in PCV#2 is 0.207 g per 100 g extract. In PCV#3 we have a
total amount of 0.339 g per 100 g extract. With the optimized protocol we were able to achieve 0.6 times
more proteinogenic amino acids per gram extract than before.