Revision as of 20:04, 21 October 2019

Project

Notebook

Week	Experiment	Participents	Description	Results
Week	Experiment	Participents	Description	Results
Week 14 04/01/2019-04/07/2019
Week 15 04/08/2019-04/21/2019
Week 16 04/15/2019-04/21/2019
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 21 05/20/2019-05/26/2019
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 23 06/03/2019-06/09/2019
Week 24 06/10/2019-06/16/2019
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 26 06/24/2019-06/30/2019
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 29 07/15/2019-07/21/2019
Week 30 07/22/2019-07/28/2019
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 32 08/05/2019-08/11/2019
Week 33 08/12/2019-08/18/2019
Week 34 08/19/2019-08/25/2019
Week 35 08/26/2019-09/01/2019
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 40 09/30/2019-10/06/2019
Week 41 10/07/2019-10/13/2019
Week 42 10/14/2019-10/20/2019

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
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
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
	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
Week 17 04/22/2019-04/28/2019	Design of the ptRNA Plasmid	Jan Müller		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
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

Vibrio

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://2019.igem.org/wiki/images/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.

Algae

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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Difference between revisions of "Team:Stuttgart/Notebook"

Revision as of 20:04, 21 October 2019

Project

Notebook

Vibrio

Vibrio - Wet lab

Vibrio - Biobrik

Vibrio - qPCR

Algae

Algae - Medium development, analysis and growth experiments

Algae - Cultvation

Algae - Disruption