iGEM 2020 — Team Aachen

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

# Team:Aachen/Notebook

iGEM Aachen - Notebook

# Our Lab Notebook

### Hitchhiker's guide to the lab! Follow our working process by going on a journey through our Labolatoral System

In our notebook, you can take a look at what we were doing in the laboratory week after week accompanied by many vivid photos. We linked our protocols so you can replicate our experiments yourselves and also get energized by M.A.R.S.

*Click on the planets to get information about the activities of the respective month*

![mars gif](https://static.igem.org/mediawiki/2020/2/23/T--Aachen--mars.gif)

June
July
August
September
October

![June](https://static.igem.org/mediawiki/2020/f/ff/T--Aachen--planet-orig.gif)

Month 1
June

![July](https://static.igem.org/mediawiki/2020/5/55/T--Aachen--planet-red.gif)

Month 2
July

![August](https://static.igem.org/mediawiki/2020/f/ff/T--Aachen--planet-green.gif)

Month 3
August

![September](https://static.igem.org/mediawiki/2020/1/17/T--Aachen--planet-blue.gif)

Month 4
September

![October](https://static.igem.org/mediawiki/2020/2/29/T--Aachen--planet-yellow.gif)

Month 5
October

[![](https://static.igem.org/mediawiki/2020/2/2b/T--Aachen--spaceship_left.png)
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#### Month 1

Week 1

Week 2

Week 3

Week 4

Week 5

Experiments

* Getting used to the equipment in the lab
* LB medium ([protocol](https://static.igem.org/mediawiki/2020/e/eb/T--Aachen--LB.pdf)) and TB medium ([protocol](https://static.igem.org/mediawiki/2020/0/01/T--Aachen--TB_Media.pdf%20)) for *Escherichia coli*

* Plasmid extraction of pET-28a from *E.coli* BL21 with Macherey-Nagel Plasmid-DNA purification kit "NucleoSpin Plasmid" (pre-cultures prepared from Lilin Feng). Concentration of plasmid measured with nanodrop: 144.7
  ng/µL
* Pre-cultures for plasmid extraction of pET-28 a from *E. coli*  BL21

* Plasmid extraction of pET-28 a from E. coli BL21 with Macherey-Nagel Plasmid-DNA purification kit “NucleoSpin Plasmid” Concentration of plasmid measured with nanodrop: 38 ng/µL
* Medium with antifoam for *Saccharomyces*([protocol](https://static.igem.org/mediawiki/2020/9/95/T--Aachen--YPG_Medium_with_adenine_and_antifoam.pdf%20))
* Cultures for anchor peptides and centrifugation of Cg-Def, LCI, MBP-1, TA2, eGFP ([protocol](https://static.igem.org/mediawiki/2020/f/fb/T--Aachen--Anchor_Cultivaton_Expression.pdf%20%20))

* Medium for *Halobacterium* ([protocol](https://static.igem.org/mediawiki/2020/f/fe/T--Aachen--Halobacterium_Medium.pdf%20)) and agar plates

* Buffer Tris 50 mM
* Cell lysis with ultrasonicator and centrifugation of anchor peptides ([protocol](https://static.igem.org/mediawiki/2020/5/5f/T--Aachen--Anchor_Cell_lysis.pdf%20)) [➤](#img004)
* Because the anchor peptides are fused to eGFP, the green/yellow colour of eGFP is visible after centrifugation
* Binding tests anchor peptides Cg-Def, LCI, MBP-1, TA2, eGFP with magnetic particles coated with polystyrene ([protocol](https://static.igem.org/mediawiki/2020/e/ee/T--Aachen--Magnet_Bindingtests.pdf%20))
* Sodium-Bicine buffer 0.2 M ([protocol](https://static.igem.org/mediawiki/2020/1/11/T--Aachen--20mM_Sodium_bicine_buffer.pdf%20)) and lipid solution 10 mM DMPC

Results

* Sequencing of pET-28a to check for restriction sites  [➤](#img001)
* Cleavage site of EcoRI found in the sequenced plasmid pET-28a   [➤](#img002)
* Cleavage site of NcoI found in the sequenced plasmid pET-28a   [➤](#img003)

* Negative control with eGFP showed no binding [➤](#img005)
* All anchor peptides showed binding to the magnetic particles, MBP-1 is the most fluorescent and therefore binds the best
* DMPC/Liposome  [➤](#img015)

### Week 2.

* Sequencing of pET-28a to check for restriction sites
* Sequence of pET-28a with cleavage sites of EcoRI and NcoI marked in red

![](https://static.igem.org/mediawiki/2020/3/37/T--Aachen--notebook1.png)

### Week 2.

* Cleavage site of EcoRI found in the sequenced plasmid pET-28a

![](https://static.igem.org/mediawiki/2020/c/c7/T--Aachen--notebook2.png)

### Week 2.

* Cleavage site of NcoI found in the sequenced plasmid pET-28a

![](https://static.igem.org/mediawiki/2020/4/46/T--Aachen--notebook3.png)

### Week 5.

* Anchor peptides after centrifugation

![](https://static.igem.org/mediawiki/2020/e/e6/T--Aachen--notebook300.jpg)
![](https://static.igem.org/mediawiki/2020/7/7b/T--Aachen--notebook4.png)

### Week 5. Result 2

* Before washing
* After washing

eGFP

LCI

MBP-1

Cg-Def

TA2

![](https://static.igem.org/mediawiki/2020/c/cc/T--Aachen--notebook5.png)
![](https://static.igem.org/mediawiki/2020/0/05/T--Aachen--notebook6.png)
![](https://static.igem.org/mediawiki/2020/4/44/T--Aachen--notebook7.png)
![](https://static.igem.org/mediawiki/2020/3/34/T--Aachen--notebook8.png)
![](https://static.igem.org/mediawiki/2020/3/33/T--Aachen--notebook9.png)
![](https://static.igem.org/mediawiki/2020/7/7d/T--Aachen--notebook10.png)
![](https://static.igem.org/mediawiki/2020/1/11/T--Aachen--notebook11.png)
![](https://static.igem.org/mediawiki/2020/8/8b/T--Aachen--notebook12.png)
![](https://static.igem.org/mediawiki/2020/2/20/T--Aachen--notebook13.png)
![](https://static.igem.org/mediawiki/2020/6/6c/T--Aachen--notebook14.png)

### Week 5. Result 3

* First try of film rehydration method with lipid 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC). DMPC in Chloroform was dried in a rotary evaporator and rehydrated in Sodium-Bicine buffer

![](https://static.igem.org/mediawiki/2020/c/c5/T--Aachen--notebook15.png)

Close

×

#### Month 2

Week 5

Week 6

Week 7

Week 8

Week 9

Experiments

* First own tries of liposome production with rehydration method ([protocol](https://static.igem.org/mediawiki/2020/c/c3/T--Aachen--SUV.pdf%20))

* Cultures of Saccharomyces cerevisiae CEN.PK113-17A
* Yeast medium
* Yeast medium YPG ([protocol](https://static.igem.org/mediawiki/2020/f/f6/T--Aachen--YPG_Medium.pdf%20))
* Tris-HCl buffer 100mM for purification and storage buffer 20 mM for mitochondria ([protocol](https://static.igem.org/mediawiki/2020/4/4a/T--Aachen--Mito_storage.pdf%20))
* Liposomes with first lipids 10 mM DMPC

* Cultures of Saccharomyces cerevisiae CEN.PK113-17A
* Yeast medium YPG ([protocol](https://static.igem.org/mediawiki/2020/f/f6/T--Aachen--YPG_Medium.pdf))
* Liposomes with rehydration method with BODIPY (protocol link) to get used to fluorescent microcopy.
* Isolation of mitochondria from  *Saccharomyces cerevisiae*  CEN.PK113-17A ([protocol](https://static.igem.org/mediawiki/2020/8/82/T--Aachen--Isolation_of_mitochondria.pdf%20)) [➤](#img016)

* LB medium and LB agar plates ([protocol](https://static.igem.org/mediawiki/2020/e/eb/T--Aachen--LB.pdf%20))
* Pre-cultures of  *Halobacterium salinarium*  and plates

* Preparation of  *Halobacterium* medium  [➤](#img017)
* Main cultures of  *H. salinarium*  ([protocol](https://static.igem.org/mediawiki/2020/6/67/T--Aachen--cultivation_of_halobaterium_salinarium.pdf%20))
* Microscopy of old DMPC liposomes from 09.07.  [➤](#img019)

Results

### Week 7.

* Isolated mitochondria from  *S. cerevisiae*

![](https://static.igem.org/mediawiki/2020/9/9d/T--Aachen--notebook16.png)

### Week 9.

* *Halobacterium salinarium* on the agar plate
* Main cultures of *H. salinarium*

![](https://static.igem.org/mediawiki/2020/4/4a/T--Aachen--notebook17.png)
![](https://static.igem.org/mediawiki/2020/9/98/T--Aachen--notebook18.png)

### Week 9.

* Liposomes still visible 3 weeks after rehydration

![](https://static.igem.org/mediawiki/2020/a/a7/T--Aachen--notebook19.png)

Close

×

#### Month 3

Week 9

Week 10

Week 11

Week 12

Week 13

Experiments

* Sucrose solutions 45%, 40%, 38%, 36%, 16%
* *Halobacterium* medium ([protocol](https://static.igem.org/mediawiki/2020/f/fe/T--Aachen--Halobacterium_Medium.pdf%20))

* Layering of sucrose solutions  [➤](#video001)
* Dry lipid film with polar soybean solution

* TE buffer for the resuspension of IDT gBlocks (short DNA, Insert) ([protocol](https://static.igem.org/mediawiki/2020/4/46/T--Aachen--TE_buffer.pdf%20%20))
* Resuspension of gblock DNA ordered from IDT (insert I1-60GFPHis) for transformation and measuring concentration with nanodrop 126.0 ng/µL; Restriction of insert I1-60GFPHis and plasmid pET-28 a with EcoRI and NcoI as well as ligation ([resuspension protocol](https://static.igem.org/mediawiki/2020/7/70/T--Aachen--Resuspension_of_IDT_g-Blocks.pdf%20))([transformation protocol](https://static.igem.org/mediawiki/2020/0/01/T--Aachen--Transformation.pdf%20))
* Centrifugation and resuspension of *H. salinarium*  [➤](#img020)
* Rehydration of soybean-lipids dry lipid film [➤](#img022)

* LB medium for  *E. coli*
* Second part of centrifugation and resuspension of  *H. salinarium*  [➤](#img023)
* Transformation of *E. coli*  C43 and *E. coli*  BL21 with last week prepared plasmid with insert (pET-28 a with I1-60GFPHis); Plates of transformed *E. colis*   and after growth on plates liquid pre-culture in tubes. Main cultures and inducing with IPTG.
* LB medium for *E. coli*  ([protocol](https://static.igem.org/mediawiki/2020/e/eb/T--Aachen--LB.pdf%20))
* Tris-HCl 20mM buffer ([protocol](https://static.igem.org/mediawiki/2020/d/d7/T--Aachen--Tris_buffer_20mM.pdf%20))
* Layering of sucrose solutions and sucrose-density-centrifugation of H. salinarium solutions for bacteriorhodopsin
* First liposome binding tests with anchors
* Precultures of  *E. coli* strains BL 21 and C 43 for each anchor peptide

* Centrifugation of  *E. coli* main cultures to find positive clones which are supposed to be green fluorescent
* TE- buffer for transformation ([protocol](https://static.igem.org/mediawiki/2020/4/46/T--Aachen--TE_buffer.pdf%20))
* 2 dry lipid films of polar-soybean-lipids
* Centrifugal filter tube for washing step in binding tests found. Test centrifugation of soybean liposomes in filter tubes destroyed the liposomes.

Results

* Sucrose-density-centrifugation of  *H. salinarium* solutions for bacteriorhodopsin did not work due to wrong rotor in centrifuge. Swinging rotor needed  [➤](#img024)
* No successful liposome binding tests due to lack of a necessary washing step of the liposomes.

* Centrifugation of  *E. coli* main cultures: no fluorescence detectable
* Centrifugal filter tube for washing step in binding tests found. Test centrifugation of soybean liposomes in filter tubes destroyed the liposomes.

### Week 10

* Video of layering of sucrose solutions

[

Your browser does not support the video tag.
](https://igemaachen.sharepoint.com/sites/iGEMAachen/Freigegebene%20Dokumente/IT%20und%20Wiki%20Subgroup/Wiki%20Texte/Labor/Lab%20notebook/layering.mp4 )

### Week 11

* Pellet after centrifuged for bacteriorhodopsin isolation
* Resuspended pellet of bacteriorhodopsin isolation

![](https://static.igem.org/mediawiki/2020/6/63/T--Aachen--notebook20.png)
![](https://static.igem.org/mediawiki/2020/f/fc/T--Aachen--notebook21.png)

### Week 11.

* Soy lipid liposomes

![](https://static.igem.org/mediawiki/2020/8/83/T--Aachen--notebook22.png)

### Week 12.

* Pellet after centrifugation for bacteriorhodopsin isolation (bacteriorhodopsin is purple)

![](https://static.igem.org/mediawiki/2020/c/c6/T--Aachen--notebook23.png)

### Week 12.

* After sucrose density centrifugation: no separation due to wrong rotor

![](https://static.igem.org/mediawiki/2020/9/99/T--Aachen--notebook24.png)

Close

×

#### Month 4

Week 14

Week 15

Week 16

Week 17

Week 18

Experiments

* Micro-extrusion of fresh soybean liposomes with 1 µm filter  [➤](#img025)

* Pre-cultures, main cultures, inducing and centrifugation of anchor peptides cgDEF, LCI, TA2, MBP-1 and eGFP for negative control ([protocol](https://static.igem.org/mediawiki/2020/0/09/T--Aachen--Proteinpurification.pdf%20))  [➤](#img027)
* Dry lipid film and rehydration of soy bean lipids with sodium-bicine buffer
* Test dialysis of soy bean lipid-liposomes with sodium-bicine buffer

* Lipid solution with new lipids 30% DOTAP, 35% DOPC and 35% PC prepared and two dry lipid films

* 30mM Tris buffer ([protocol](https://static.igem.org/mediawiki/2020/7/76/T--Aachen--Tris_buffer_30mM.pdf%20))
* Try liposomes with new lipids: 30% DOTAP, 35% DOPC and 35% PC in 30mM Tris buffer ([protocol](https://static.igem.org/mediawiki/2020/c/c3/T--Aachen--SUV.pdf%20))
* First try of the positive control for the CAR Reaction in Jülich: didn´t work. We also prepared the stock solutions for more reactions

* 1M sorbitol buffer (protocol)
* Second try of liposome formation with new lipids: 30% DOTAP, 35% DOPC and 35% PC with 1M sorbitol buffer
* Liposomes with polar-soybean-lipids and stained with nil red [➤](#img030)
* Testing the positive control at FZ Jülich of the CAR reaction. ([protocol](https://static.igem.org/mediawiki/2020/d/d7/T--Aachen--In_vitro_approach_carboxylate_reductase_j%C3%BClich.pdf%20))

Results

* After 48 hours of dialysis were less but still a few liposomes visible  [➤](#img028)

* Try liposomes with new lipids: no liposome formation
* First try of the positive control for the CAR Reaction in Jülich: didn´t work.

* Second try of liposomes with new lipids: no liposome formation
* Testing the positive control at FZ Jülich of the CAR reaction: results for the substrate concentration weren´t as expected.

### Week 14.

* Micro-extrusion of fresh soybean liposomes

Before

After

![](https://static.igem.org/mediawiki/2020/2/2d/T--Aachen--notebook25.png)
![](https://static.igem.org/mediawiki/2020/9/9f/T--Aachen--notebook26.png)

### Week 15.

* MBP-1 anchor peptide fused to eGFP and eGFP as negative control after centrifugation. Green/Yellow colour due to eGFP

![](https://static.igem.org/mediawiki/2020/3/33/T--Aachen--notebook27.png)

### Week 15.

* After 48 hours of est dialysis of soy bean lipid-liposomes with sodium-bicine buffer were less but still a few liposomes visible

Before

After

![](https://static.igem.org/mediawiki/2020/0/0a/T--Aachen--notebook28.png)
![](https://static.igem.org/mediawiki/2020/a/a5/T--Aachen--notebook29.png)

### Week 17.

* Microscope pictures of soybean-liposomes stained with nile red

![](https://static.igem.org/mediawiki/2020/8/8a/T--Aachen--notebook30.png)

Close

×

#### Month 5

Week 18

Week 19

Week 20

Week 21

Week 22

Experiments

* New lipid mixture of 30% DOTAP, 35% DOPC and 35% PC rehydrated with 1M Sorbitol buffer and liposome formation
* Dilution of lipid mixture (1:10 and 1:100) and liposome formation
* Liposomes with only one of the lipids each: DOTAP, DOPC and POPC  [➤](#img031)
* Transformation of  *E. coli* BL21 with new ordered plasmid with I1-60GFPHis (orderd from Twist Bioscience). No Restriction needed, only steps … in the corresponding protocol. Resuspension of the DNA with 120 µL TE buffer. After transformation growth on plates ([protocol](https://static.igem.org/mediawiki/2020/0/01/T--Aachen--Transformation.pdf%20))
* Dry lipid film of POPC-lipids

* Cultures of transformed  *E. coli* s with I1-60FPHis from plates to liquid cultures, inducing of main cultures with IPTG and centrifugation  [➤](#img034)
* Phosphate-solution and solubilisation buffer ([protocol](https://static.igem.org/mediawiki/2020/5/55/T--Aachen--Solubilisation_buffer.pdf%20))
* TB medium for  *E. coli*  ([protocol](https://static.igem.org/mediawiki/2020/0/01/T--Aachen--TB_Media.pdf%20%20))
* Liposome binding tests with anchor peptides LCI, cgDef and eGFP as negative control. Used buffer was 30mM Tris ([protocol](https://static.igem.org/mediawiki/2020/2/22/T--Aachen--Liposome_Bindingtests.pdf%20))  [➤](#img)
* Dry lipid films with POPC lipids and rehydration
* Whole cell approach at FZ Jülich for CAR reaction. ([protocol](https://static.igem.org/mediawiki/2020/6/6b/T--Aachen--Whole_Cell_Approach_Proof_Juelich.pdf%20)) .
* Calibration for the alcohol in the HPLC
* Positive control again ([protocol](https://static.igem.org/mediawiki/2020/d/d7/T--Aachen--In_vitro_approach_carboxylate_reductase_j%C3%BClich.pdf%20))

* Dialysis of PC liposomes with 0,5 mM pyranin  [➤](#img035)
* Rehydration of POPC dry lipid film with 30 mM Tris-HCl buffer with 0.5 mM pyranin. [➤](#img046)
* Binding tests with anchor peptides and POPC liposomes. Different concentrations of anchor peptides tried: 1 µM, 0.5 µM, 0.1 µM, 0.05 µM, 0.01 µM
* Positive control for the CAR reaction at FZ Jülich ([protocol](https://static.igem.org/mediawiki/2020/d/d7/T--Aachen--In_vitro_approach_carboxylate_reductase_j%C3%BClich.pdf%20)). Data was as expected.
* Liposome formation with a lipid batch of 80 µL POPC, 20 µL DOTAP and 20 µL of the fluorescent labeled DOPE lipid ([protocol](https://static.igem.org/mediawiki/2020/d/d7/T--Aachen--In_vitro_approach_carboxylate_reductase_j%C3%BClich.pdf%20)) [➤](#img050)

* PBS buffer pH 7.4, 10x PBS buffer ([protocol](https://static.igem.org/mediawiki/2020/b/b9/T--Aachen--PBS_%28Phosphate_Buffered_Saline%29.pdf%20))and Stripping buffer pH 2 for gel electrophoresis
* Two mini gels: Separation gel and stacking gel ([protocol](https://static.igem.org/mediawiki/2020/3/35/T--Aachen--10x_SDS_electrophoresis_buffer.pdf%20)), storage in wet environment for next week at 4°C.
* Watched microfluidics with liposomes done by Volkan
* Dry lipid film and rehydration of DOTAP lipids with Tris-HCL 30 mM pH 7.4
* Binding tests with DOTAP liposomes with strep-eGFP,strep-LCI and Met rich
* Protein purification of the anchor peptides TA2 and cg-Def at FZ Jülich ([protocol](https://static.igem.org/mediawiki/2020/0/09/T--Aachen--Proteinpurification.pdf%20)). Preparation of buffer for purification (equilibration, washing, elution and desalting buffer) ([equilibration protocol](https://static.igem.org/mediawiki/2020/4/42/T--Aachen--Equilibration_50mM_Tris.pdf%20))
  ([elution protocol](https://static.igem.org/mediawiki/2020/7/72/T--Aachen--Elutionbuffer_50mM_Tris.pdf%20%20))([desalting protocol](https://static.igem.org/mediawiki/2020/9/9e/T--Aachen--Desalting_10mM.pdf%20)).
* Liposome formation of a lipid batch with 100 µL POPC. Binding tests with anker peptides TA2, cg-Def, Cecropin A, Maquace, eGFP and LCI ([protocol](https://static.igem.org/mediawiki/2020/2/22/T--Aachen--Liposome_Bindingtests.pdf%20))

Results

* New lipid mixture: no liposomes visible
* Dilution of lipid mixture: no liposomes visible

* Cultures of transformed *E. colis* with I1-60GFPHis from plates to liquid cultures, inducing of main cultures with IPTG and centrifugation: no fluorescence visible after centrifugation; made a new kryostock of transformed *E. coli*
* *E. coli* carry the plasmid, because they are able to growth on the plates with kanamycin antibiotic (the plasmid contains a kanamycin resistance)
* Liposome binding tests with anchor peptides:Anchors cannot get through the dialysis tube, no fluorescence outside the tube but still inside, even if no liposomes are added
* GUV formation with POPC liposomes and Sorbitol 1 M buffer ([protocol](https://static.igem.org/mediawiki/2020/0/09/T--Aachen--GUV.pdf%20)), GUV were visible and there were more bigger liposomes  [➤](#img044)
* Data for the whole cell approach was as expected.
* Data for the positive control of the CAR reaction still not as expected.

* After the liposomes were rehydrated with the fluorescent dye pyranine they appeared as dark spots under the fluorescence microscope  [➤](#img046)
* Dilution of lipid mixture: no liposomes visible
* After the dialysis step which should remove pyranine from the outer phase the liposomes still appeared darker than the background. This could be because the pH of the buffer increased during the dialysis for unknown reasons from 7.4 to 7.76 and the fluorescence of pyranine is highly pH sensitive
* No fluorescence visible with 0.1 µM, 0.05 µM, 0.01 µM anchor peptides
* Too much fluorescence visible at 1 µM anchor peptides
* Liposomes with fluorescence visible with 0.5 µM
* We experienced some difficulties with liposome formation. Probably our lipids got too old or maybe oxidized.

* Binding tests: no liposomes visible after incubation for binding. Try new tests with less shaking.
* Protein purification of the anchor peptides TA2 and cg-Def at FZ Jülich: concentration of TA2 0.164 mg/mL and for cg-Def 0.163 mg/mL

### Week 18.

* Liposomes with only one of the lipids each: DOTAP, DOPC and POPC
* No liposomes visible

DOTAP

DOPC

PC

![](https://static.igem.org/mediawiki/2020/8/8f/T--Aachen--notebook31.png)
![](https://static.igem.org/mediawiki/2020/5/57/T--Aachen--notebook32.png)
![](https://static.igem.org/mediawiki/2020/b/bf/T--Aachen--notebook33.png)

### Week 19.

* Agar plate with transformed  *E. coli* s with the I1-60GFP His insert

![](https://static.igem.org/mediawiki/2020/4/45/T--Aachen--notebook34.png)

### Week 19.

* GUV´s with POPC lipids stained with nil red

![](https://static.igem.org/mediawiki/2020/6/64/T--Aachen--notebook44.jpg)
![](https://static.igem.org/mediawiki/2020/0/0d/T--Aachen--notebook45.jpg)

### Week 20.

* Dialysis of PC liposomes with 0,5 mM pyranin

Before

After

![](https://static.igem.org/mediawiki/2020/f/fc/T--Aachen--notebook35.png)
![](https://static.igem.org/mediawiki/2020/3/3d/T--Aachen--notebook36.png)
![](https://static.igem.org/mediawiki/2020/1/15/T--Aachen--notebook37.png)

### Week 20.

* In Brightfield mode
* In GFP mode (exposure time 781 µs, gain 5.5 dB)

![](https://static.igem.org/mediawiki/2020/8/8f/T--Aachen--Pyranin_Brightfield.jpg)
![](https://static.igem.org/mediawiki/2020/0/04/T--Aachen--Pyranin_eGFP.jpg )

### Week 20.

* Liposomes were visible (picture 1) and they showed a higher fluorescence than the background (picture 2)
* With this results more tests should be performed with concentrations of 0.4 µM, 0.5 µM and 0.6 µM

![](https://static.igem.org/mediawiki/2020/c/cd/T--Aachen--LCI_BF.png)
![](https://static.igem.org/mediawiki/2020/2/29/T--Aachen--mbp.jpeg)

### Week 20.

* Liposomes under the confocal microscope

![](https://static.igem.org/mediawiki/2020/2/2e/T--Aachen--DOTAP_Confocal.png)

### Week 21.

* Pictures of binding tests with every anchor peptide with a concentration of 0.5 µM
* Brightfield mode
* eGFP mode

eGFP

LCI

Met Rich

Cecropin A

MA

![](https://static.igem.org/mediawiki/2020/6/6a/T--Aachen--eGFP%2C_strep.jpg )
![](https://static.igem.org/mediawiki/2020/5/52/T--Aachen--LcI%2C_strep.jpg)
![](https://static.igem.org/mediawiki/2020/7/70/T--Aachen--met_rich%2C_his.jpg)
![](https://static.igem.org/mediawiki/2020/d/d1/T--Aachen--cecropin%2C_his.jpg)
![](https://static.igem.org/mediawiki/2020/d/de/T--Aachen--MA%2C_his.jpg)
![](https://static.igem.org/mediawiki/2020/d/d2/T--Aachen--eGFP%2C_strep%2C_dark.jpg)
![](https://static.igem.org/mediawiki/2020/6/64/T--Aachen--LcI%2C_strep%2C_dark.jpg )
![](https://static.igem.org/mediawiki/2020/6/6d/T--Aachen--met_rich%2C_his%2C_dark.jpg )
![](https://static.igem.org/mediawiki/2020/6/61/T--Aachen--cecropin%2C_his%2C_dark.jpg )
![](https://static.igem.org/mediawiki/2020/e/ee/T--Aachen--MA%2C_his%2C_dark.jpg )

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# Team:Aachen/Engineering

iGEM Aachen - Engineering Success

# Engineering Success

### A small step for us, a giant leap for M.A.R.S.

In our project, we realized that some of the experiments we planned did not work within the first try. Therefore, we had to evaluate the problems we faced and had to come up with new solutions. Even if something works, there is always the opportunity to improve further experiments.

![mars gif](https://static.igem.org/mediawiki/2020/2/23/T--Aachen--mars.gif)

Search for the anchor peptides

![](https://static.igem.org/mediawiki/2020/2/2e/T--Aachen--DOTAP_Confocal.png)
One of our project's central laboratory goal was to find an anchor peptide that binds to the magnetic particle as well as a second anchor peptide that binds to our liposomes.

So, we first had to synthesize liposomes. After some research, we decided to use a mixture of PC, DOTAP, and DOPC to have slightly positively charged liposomes for better bacteriorhodopsin integration[**[1]**](#ref). The desired mixture of 30% DOTAP, 35% DOPC, and 35% POPC did not work; there were no liposomes visible. We then tried liposomes consisting of only POPC first. POPC worked with different buffers Tris-HCl 30 mM, pH 7.4([protocol](https://static.igem.org/mediawiki/2020/7/76/T--Aachen--Tris_buffer_30mM.pdf)) and Sorbitol 1 M([protocol](https://static.igem.org/mediawiki/2020/7/7c/T--Aachen--1M_Sorbitol_buffer.pdf)) . DOTAP and DOPC also worked for themselves with Tris-HCl 30 mM, pH 7.4([protocol](https://static.igem.org/mediawiki/2020/7/76/T--Aachen--Tris_buffer_30mM.pdf)) . After that, we tried a mixture of 80% POPC and 20% DOTAP to get closer to the desired positively charged composition, which also worked.

![](https://static.igem.org/mediawiki/2020/c/cd/T--Aachen--LCI_BF.png)
![](https://static.igem.org/mediawiki/2020/2/29/T--Aachen--mbp.jpeg)

With the formed liposomes, we could start the binding tests with different anchor peptides. First binding tests were performed with POPC liposomes because the formation of these was the most reliable. Our first approach was to try centrifugation through a molecular filter in order to separate liposomes from unbound anchors. But the centrifugation seemed to destroy the liposomes, which made us think about alternatives.

Another idea was to try size exclusion chromatography. This method has the risk that the liposomes interact with the column material and break. We therefore decided to try out a gentler approach. With dialysis, we could keep the bigger liposomes inside the dialysis tube and lower the concentration of unbound anchors because they are small enough to get through the dialysis tube. Due to the anchor peptides' size at around 35 kDa (anchor peptide + eGFP), we tried a dialysis tube with a molecular cut off 50 kDa. After a few tries, we had to realize that the anchors could not get through the tube membrane. Evaluating the problem, we concluded that they might create a hydrate shell, making them way bigger. New tests should be performed with a molecular cut off 100 kDa or even 300 kDa.

Due to the limited time left in the lab, we decided to first look for the best binding candidate with just qualitative tests. With the most promising candidates further tests with dialysis tubes should be performed. The new tests were performed with low anchor concentrations to see if the liposomes or the whole background would show fluorescence first. So far only LCI showed a fluorescence of the liposomes. Therefore, more tests with LCI should be performed.

First results

The first test with the magnetic particles turned out well. We identified MBP-1 to have the best binding due to the highest fluorescence. Under the same conditions a higher fluorescence means that more anchor peptides are bound to the target surface.

eGFP (negative control)

LCI

MBP-1

Cg-Def

TA2

before washing

![](https://static.igem.org/mediawiki/2020/c/cc/T--Aachen--notebook5.png)

![](https://static.igem.org/mediawiki/2020/0/05/T--Aachen--notebook6.png)

![](https://static.igem.org/mediawiki/2020/4/44/T--Aachen--notebook7.png)

![](https://static.igem.org/mediawiki/2020/3/34/T--Aachen--notebook8.png)

![](https://static.igem.org/mediawiki/2020/3/33/T--Aachen--notebook9.png)

after washing

![](https://static.igem.org/mediawiki/2020/7/7d/T--Aachen--notebook10.png)

![](https://static.igem.org/mediawiki/2020/1/11/T--Aachen--notebook11.png)

![](https://static.igem.org/mediawiki/2020/8/8b/T--Aachen--notebook12.png)

![](https://static.igem.org/mediawiki/2020/2/20/T--Aachen--notebook13.png)

![](https://static.igem.org/mediawiki/2020/6/6c/T--Aachen--notebook14.png)

With these first results we concluded that a possible biadhesive peptide would be a fusion protein of MBP-1 and LCI. Both anchor peptides are connected through a stiff spacer Domain Z. This construct is able to connect polystyrene coated magnetic particles to POPC liposomes. Further test should be performed with different lipid compositions, especially with the desired slightly positively charged lipids. The whole fusion protein can then be expressed and binding of both complexes can be checked. This could be done through the use of common bar magnets to see if the liposomes would also be separated.

As an outlook for further experiments, it would be recommendable to find an anchor that binds to the magnetic particle but not to the liposomes and an anchor peptide that binds the liposomes but not the magnetic particle. This can ensure that only liposomes and magnetic particles are connected and not two magnetic particles or two liposomes with each other. Therefore, experiments should be performed with the chosen anchor peptides and both of the target surfaces. This experiment can be considered successful if, and only if, each anchor peptide binds only to its expected target surface.

### References

**[1] Ritzmann N, Thoma J, Hirschi S, Kalbermatter D, Fotiadis D, Müller DJ. Fusion Domains Guide the Oriented Insertion of Light-Driven Proton Pumps into Liposomes. Biophys J. 2017 Sep 19;113(6):1181-1186. doi: 10.1016/j.bpj.2017.06.022. Epub 2017 Jul 8. PMID: 28697898; PMCID: PMC5607040**

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

# Team:Aachen/Experiments

iGEM Aachen - Experiments

# Experiments in the Lab

### Here's how the magic happened! In the [Institute of Biotechnology](#Schwanelab) and the [FZ Jülich](#FZJueLab)

During the lockdown in 2020, we researched and planned our experiments bit by bit. Entering the laboratory in June, we were able to present well-developed and -organized plans for the upcoming weeks. It enabled us to carry out most of our
planned experiments, despite the restriction to only one permitted person at a time.

Due to the possibility of working at two different locations, namely the [Institute of Biotechnology](#Schwanelab) and [FZ
Jülich](#FZJueLab), we could gain a lot of experiences and results, both for each of us personally, as well as specifically as part of our project.
As the result of the fruitful work in the lab we collected quite a [Library of Protocols](#Library) of all experiments we conducted in the scope of the project M.A.R.S. On this page you
have access to all our lab protocols as well as comprehensive [Results](#Results).

![mars gif](https://static.igem.org/mediawiki/2020/2/23/T--Aachen--mars.gif)

Institute of Biotechnology

Our experiments in the laboratory of Prof. Dr. Schwaneberg can be divided into four different categories. The binding tests of our anchor peptides on various surfaces to construct and express an optimal binding complex, the liposome
formation and the protein expression and lastly the protein integration of our needed proteins.

Concerning the binding tests, we want use different antimicrobial peptides and test their binding abilities on our polystyrene coated magnetic particles to determine the best one. We would then use this one as the magnet-binding domain in
our binding complex. To evaluate the binding abilities of the different binding domains, we first express different anchor peptides with an eGFP label. We then check the remaining fluorescence under a confocal microscope after several
washing steps. To determine the polymersome or liposome binding domain we proceed analogously. After finding the best binding peptides, we can express a biadhesive binding complex using both domains and a stiff linker domain to connect
our ATP factories to our magnetic particles.

We have searched for purification methods for bacteriorhodopsin from *Halobacterium salinarium* and ATP synthase from *Saccharomyces cerevisiae*. For the ATP synthase we want to design a plasmid with the sequence of the first 60
amino acids of an ATP synthase inhibitor protein. With this protein an affinity purification of the ATP synthase from the isolated mitochondria can be performed.

For the lipid formation we decided to use the film rehydration technique to rehydrate a dried lipid film and create small unilamellar vesicles. To make the liposomes easier to detect under the microscope we want to form giant unilamellar
vesicles. This was achieved by creating an oil-surfactant mix where an aqueous solution, which contains the lipid batch, is added [**[1]**](#ref). The spontaneous formation of
surfactant-stabilized water-in-oil droplets with a spherical supported lipid bilayer at their periphery is then induced by manual shaking and the average diameter is determined by the shaking duration and intensity. A small amount of
ATTO-labelled lipids where included in the lipid batch to confirm the liposome formation using fluorescence microscopy.

To finally integrate our chosen proteins, we want to start with integration of bacteriorhodopsin into the giant unilamellar vesicles. Therefore, we first solubilize the purple membrane. In order to integrate the membrane proteins, it is
necessary to destabilize the liposome membrane using the detergence Triton-X 100. We plan to add Triton-X-100 together with our bacteriorhodopsin to the solution and to incubate the mixture to perform the integration. For detergence
removal we plan to perform multiple washing steps using bio beads. For a confirmation of the correct integration we intended to use pyranine which is a pH indicating fluorescent dye. Through the acidification of the inner phase of the
vesicles by bacteriorhodopsin the pyranine inside the vesicles would appear darker than their background. Thus, we would be able to visibly proof the correct integration using fluorescent microscopy. The destabilisation method could also
work to integrate the ATP synthase and therefore create our final chassis. To proof the successful integration of bacteriorhodopsin and the ATP synthase together we would test our system in the context of a real industrial application at
the FZ Jülich and model it to generate theoretical data.

FZ Jülich

At the FZ Jülich we perform multiple tests on a carboxylate reductase from the organism *Nocardia otitidiscaviarum*. This enzyme is capable of converting 3-hydroxybenzoic acid to 3-hydroxybenzaldehyde. Therefore, we prepare triplets
of reaction vials where we add the carboxylate reductase, 3-hydroxybenzoic acid as the substrate, ATP, NADH, polyphosphate, the needed enzyme to perform the conventional ATP regeneration and a 4x concentrated MOPS buffer containing
glucose and MgCl2. With this method kinases are used to fuse phosphate groups from polyphosphates to ADP. This performs the ATP regeneration[**[2]**](#ref). After getting comfortable with the
procedures, we want to show using negative controls without ATP, that the lack of ATP leads to no substrate conversion at all, which means that ATP is crucial for that enzyme. Another negative control without the enzymes which normally
performs the ATP regeneration will be put to test, to show the importance of a cofactor regeneration system.

To get more insight into the functionality of that enzyme, we also want to monitor the product conversion of a whole cell approach where the carboxylate reductase is expressed in *E. coli* K12 cells which tolerate the aldehyde
product. Using these different attempts, we can collect valuable data which helps us to compare our system to the ATP regeneration approaches above. Therefore, our last testing study is supposed to focus on using our ATP-factories as the
needed cofactor regeneration system. Furthermore, we simulated how M.A.R.S. would act in this reaction setup to additionally generate data from a bioinformatic point of view. This can be seen on our modeling page. A successful integration
of the bacteriorhodopsin and ATP synthase would result in a detectable amount of product conversion.

*Push the buttons to view the protocols from each category*

Buffer
and
Media

Proteins
and
Integration

Liposomes
and
Binding

Proof
of
Concept

![](https://static.igem.org/mediawiki/2020/8/80/T--Aachen--RU.png)

![](https://static.igem.org/mediawiki/2020/b/b6/T--Aachen--GU.png)

![](https://static.igem.org/mediawiki/2020/c/ce/T--Aachen--BU.png)

![](https://static.igem.org/mediawiki/2020/b/bf/T--Aachen--YU.png)

[Halobacterium medium](https://static.igem.org/mediawiki/2020/f/fe/T--Aachen--Halobacterium_Medium.pdf)

[KPi buffer](https://static.igem.org/mediawiki/2020/6/62/T--Aachen--400mM_KPi.pdf%20)

[TB media](https://static.igem.org/mediawiki/2020/0/01/T--Aachen--TB_Media.pdf%20)

[Mitochondria storage buffer](https://static.igem.org/mediawiki/2020/4/4a/T--Aachen--Mito_storage.pdf%20)

[Sodium-bicine buffer](https://static.igem.org/mediawiki/2020/1/11/T--Aachen--20mM_Sodium_bicine_buffer.pdf%20)

[Sorbitol buffer](https://static.igem.org/mediawiki/2020/7/7c/T--Aachen--1M_Sorbitol_buffer.pdf%20%20)

[Mitochondria washing buffer](https://static.igem.org/mediawiki/2020/9/9e/T--Aachen--Mito_washing.pdf%20)

[YPG medium with adenine and antifoam](https://static.igem.org/mediawiki/2020/9/95/T--Aachen--YPG_Medium_with_adenine_and_antifoam.pdf)

[Solubilisation buffer](https://static.igem.org/mediawiki/2020/5/55/T--Aachen--Solubilisation_buffer.pdf%20)

[YPG Medium](https://static.igem.org/mediawiki/2020/f/f6/T--Aachen--YPG_Medium.pdf)

[TE buffer](https://static.igem.org/mediawiki/2020/4/46/T--Aachen--TE_buffer.pdf%20)

[Tris-HCl buffer
20 mM](https://static.igem.org/mediawiki/2020/d/d7/T--Aachen--Tris_buffer_20mM.pdf%20)

[Tris-HCl buffer
 30 mM](https://static.igem.org/mediawiki/2020/7/76/T--Aachen--Tris_buffer_30mM.pdf%20)

[Phosphate buffer 1 M](https://static.igem.org/mediawiki/2020/6/6e/T--Aachen--Phosphate_buffer_1M_pH7.pdf%20)

[Washing buffer 50 mM Tris](https://static.igem.org/mediawiki/2020/e/e9/T--Aachen--Washing_buffer_50mM_tris_pH_7.5.pdf%20)

[Tris-HCl buffer 50 mM](https://static.igem.org/mediawiki/2020/e/e2/T--Aachen--Tris_buffer_50mM.pdf%20)

[4x MOPS buffer](https://static.igem.org/mediawiki/2020/e/ee/T--Aachen--4x_Mops_Buffer.pdf%20%20)

[10x SDS electrophoresis buffer](https://static.igem.org/mediawiki/2020/3/35/T--Aachen--10x_SDS_electrophoresis_buffer.pdf%20)

[Phosphate buffered Saline](https://static.igem.org/mediawiki/2020/b/b9/T--Aachen--PBS_%28Phosphate_Buffered_Saline%29.pdf%20%20)

[SDS 1x loading buffer](https://static.igem.org/mediawiki/2020/6/60/T--Aachen--SDS_1x_Loading_Buffer.pdf%20%20)

[Desalting buffer 10 mM Tris](https://static.igem.org/mediawiki/2020/9/9e/T--Aachen--Desalting_10mM.pdf%20%20)

[Equilibration buffer 50 mM Tris](https://static.igem.org/mediawiki/2020/4/42/T--Aachen--Equilibration_50mM_Tris.pdf%20)

[LB medium](https://static.igem.org/mediawiki/2020/e/eb/T--Aachen--LB.pdf%20%20)

[Elution buffer 50 mM Tris](https://static.igem.org/mediawiki/2020/7/72/T--Aachen--Elutionbuffer_50mM_Tris.pdf%20%20)

[Bacteriorhodopsin integration 1](https://static.igem.org/mediawiki/2020/2/25/T--Aachen--br_integration1.pdf%20%20%20)

[Bacteriorhodopsin integration 2](https://static.igem.org/mediawiki/2020/6/62/T--Aachen--br_integration2.pdf%20%20)

[Bacteriorhodopsin integration 3](https://static.igem.org/mediawiki/2020/3/34/T--Aachen--br_integration3.pdf%20%20)

[Bacteriorhodopsin integration 4](https://static.igem.org/mediawiki/2020/e/e6/T--Aachen--br_integration4.pdf%20%20)

[Immuno-Detection](https://static.igem.org/mediawiki/2020/d/dd/T--Aachen--Immuno-Detection.pdf%20%20%20)

[Insert digest](https://static.igem.org/mediawiki/2020/e/e2/T--Aachen--Insert_digest.pdf%20%20)

[Plasmid digest](https://static.igem.org/mediawiki/2020/5/5a/T--Aachen--Plasmid_digest.pdf%20%20)

[Plasmid ligation](https://static.igem.org/mediawiki/2020/e/e3/T--Aachen--Plasmid_ligation.pdf%20)

[Cultivation of Halobaterium salinarium](https://static.igem.org/mediawiki/2020/6/67/T--Aachen--cultivation_of_halobaterium_salinarium.pdf%20%20)

[Purification of the Purplemembrane of Halobaerium salinarium](https://static.igem.org/mediawiki/2020/a/ac/T--Aachen--Purification_of_the_Purple_Membrane_%28PM%29_from_Halobacterium_salinarum.pdf%20%20)

[Resuspension of IDT g\_Blocks](https://static.igem.org/mediawiki/2020/7/70/T--Aachen--Resuspension_of_IDT_g-Blocks.pdf%20)

[SDS Gel-Elektrophoresis](https://static.igem.org/mediawiki/2020/d/de/T--Aachen--SDS_Gel-Electrophoresis_%28SDS-PAGE%29.pdf%20%20)

[Transformation](https://static.igem.org/mediawiki/2020/0/01/T--Aachen--Transformation.pdf%20)

[Western Blot](https://static.igem.org/mediawiki/2020/b/b6/T--Aachen--Western_Blot.pdf%20)

[Isolation of Mitochondria](https://static.igem.org/mediawiki/2020/8/82/T--Aachen--Isolation_of_mitochondria.pdf%20)

[Anchor cultivation and expression](https://static.igem.org/mediawiki/2020/f/fb/T--Aachen--Anchor_Cultivaton_Expression.pdf%20%20)

[Proteinpurification for anchor peptides](https://static.igem.org/mediawiki/2020/0/09/T--Aachen--Proteinpurification.pdf%20%20)

[Anchor cell lysis](https://static.igem.org/mediawiki/2020/5/5f/T--Aachen--Anchor_Cell_lysis.pdf%20)

[SUV/normal liposome formation](https://static.igem.org/mediawiki/2020/c/c3/T--Aachen--SUV.pdf%20%20)

[GUVs](https://static.igem.org/mediawiki/2020/0/09/T--Aachen--GUV.pdf%20)

[Magnet binding tests](https://static.igem.org/mediawiki/2020/e/ee/T--Aachen--Magnet_Bindingtests.pdf%20%20)

[Liposome Binding tests](https://static.igem.org/mediawiki/2020/2/22/T--Aachen--Liposome_Bindingtests.pdf%20)

[In vitro approach carboxylate reductase](https://static.igem.org/mediawiki/2020/d/d7/T--Aachen--In_vitro_approach_carboxylate_reductase_j%C3%BClich.pdf%20%20)

[Whole cell approach](https://static.igem.org/mediawiki/2020/6/6b/T--Aachen--Whole_Cell_Approach_Proof_Juelich.pdf%20)

Results

Here you will find the entirety of our lab results (failures included). These consist of:

1. Liposome formation
2. Liposome and magnetic particle binding tests
3. Protein purification and integration
4. Application in a relevant context

For further information about the experiments please refer to our [Lab Notebook](https://2020.igem.org/Team%3AAachen/Notebook)

1 The first result we achieved was the creation of liposomes with different lipid compositions.
For the first tries, we also used a soybean polar lipid mixture.
Our finally desired lipids would be slightly positive to enable better protein integration.
It is proven that bacteriorhodopsin can be integrated easier into positively charged liposomes due to their low negative charge. [**[3]**](#ref)

The compositions that worked are:

Soybean polar lipid mixture with 0.2 M sodium-bicine buffer

DOTAP with Tris 30mM buffer

DOPC with Tris 30mM buffer

POPC with Tris 30mM buffer

20% DOTAP, 80% POPC with Tris 30 mM buffer with atto-labled lipids

![](https://static.igem.org/mediawiki/2020/2/2d/T--Aachen--notebook25.png)

![](https://static.igem.org/mediawiki/2020/8/8f/T--Aachen--notebook31.png)

![](https://static.igem.org/mediawiki/2020/5/57/T--Aachen--notebook32.png)

![](https://static.igem.org/mediawiki/2020/b/bf/T--Aachen--notebook33.png)

![](https://static.igem.org/mediawiki/2020/2/2e/T--Aachen--DOTAP_Confocal.png)

2 As a second result, we were looking for both anchor peptides. One needs to bind the polystyrene coated magnetic particles, and the other one should bind to the liposome. The anchor peptides are fused to eGFP to make them visible under the microscope.

Higher fluorescence means more anchors are bound to the magnetic particle´s surface. Within the first test with the magnetic particles, we identified MBP-1 to have the best binding. Under same conditions, MBP-1 had the highest fluorescence.

eGFP (negative control)

LCI

MBP-1

Cg-Def

TA2

before washing

![](https://static.igem.org/mediawiki/2020/c/cc/T--Aachen--notebook5.png)

![](https://static.igem.org/mediawiki/2020/0/05/T--Aachen--notebook6.png)

![](https://static.igem.org/mediawiki/2020/4/44/T--Aachen--notebook7.png)

![](https://static.igem.org/mediawiki/2020/3/34/T--Aachen--notebook8.png)

![](https://static.igem.org/mediawiki/2020/3/33/T--Aachen--notebook9.png)

after washing

![](https://static.igem.org/mediawiki/2020/7/7d/T--Aachen--notebook10.png)

![](https://static.igem.org/mediawiki/2020/1/11/T--Aachen--notebook11.png)

![](https://static.igem.org/mediawiki/2020/8/8b/T--Aachen--notebook12.png)

![](https://static.igem.org/mediawiki/2020/2/20/T--Aachen--notebook13.png)

![](https://static.igem.org/mediawiki/2020/6/6c/T--Aachen--notebook14.png)

The tests with the liposomes were more difficult. There is no easy way of separating the liposomes from unbound anchors. We first tried centrifugation, which did not work because the liposomes broke. The next try was to use a gentler method for separation, dialysis. Due to the bigger size of the anchors, we tried a molecular cut off 50 kDa. The liposomes were stable this time, but the anchors did not get through the dialysis tube. Next tries should be performed with a molecular cut off 100 kDa and maybe even 300 kDa.

Because we could not get bigger dialysis tubes fast enough, we tried out our binding candidates with low concentrations. The aim was to see whether the liposomes or the whole background shows fluorescence first.

POPC with Tris 30mM buffer, 0.2 µM eGFP, Brightfield mode

POPC with Tris 30mM buffer, 0.2 µM eGFP, eGFP mode

POPC with Tris 30mM buffer, 0.25 µM LcI, eGFP mode

POPC with Tris 30mM buffer, 0.25 µM LcI, eGFP mode with zoom

![](https://static.igem.org/mediawiki/2020/f/f3/T--Aachen--result1.jpg)

![](https://static.igem.org/mediawiki/2020/b/b8/T--Aachen--result2.jpg)

![](https://static.igem.org/mediawiki/2020/5/50/T--Aachen--result3.jpg)

![](https://static.igem.org/mediawiki/2020/6/62/T--Aachen--result4.jpg)

With these first results we concluded that a possible biadhesive peptide would be a fusion protein of MBP-1 and LCI. Both anchor peptides are connected through a stiff spacer Domain Z. This construct can connect polystyrene coated magnetic particles to POPC liposomes. Further test should be performed with different lipid compositions, especially with the desired slightly positively charged lipids. The whole fusion protein can then be expressed and binding of both complexes can be checked. This could be done through magnetically separation to see if the liposomes would also be separated.

3 For the protein purification we developed a plan for the bacteriorhodopsin purification. We purified it from *Halobacterium salinarium* and were able to see the purple membrane that includes the bacteriorhodopsin. The purple membrane can be seen in purple in the following picture.
![](https://static.igem.org/mediawiki/2020/c/c6/T--Aachen--notebook23.png)

The bacteriorhodopsin can be separated from other cell components through sucrose density gradient centrifugation. Due to an insufficient rotor angle in the centrifuge a clear separation was not possible. A swing rotor is needed to perform a successful separation. With bought bacteriorhodopsin we also developed a plan for the integration in liposomes. We therefore tried the solubilisation and created a detailed plan for validation of effective.

Concerning the ATP synthase, we first purified mitochondria from yeast cells. According to "The affinity purification and characterization of ATP synthase complexes from mitochondria" [**[4]**](#ref) we designed a plasmid with the sequence of the first 60 amino acids of an ATP synthase inhibitor protein (BBa\_K3456000). We expressed this part of the inhibitor with an eGFP and its tag. There was no fluorescence visible in the samples of the centrifuged cells. Therefore, tests need to be performed to validate that the right protein was expressed. This test could be a gel electrophoresis to check the right size. Due to its his tag the protein can be purified through affinity purification with a nickel coated sepharose column.

The inhibitor protein can then bind to the ATP synthase from yeast mitochondria and the whole complex can again be purified through affinity purification with a nickel coated sepharose column. The isolated mitochondria from *Saccharomyces cerevisiae* including ATP synthase were validated with antibodies through a western blot.

4 We collected data from multiple reactions of the carboxylate reductase in the FZ Jülich. Changing different parameters concerning the ATP regeneration could give us a special insight on how ATP affects this kind of reaction. Furthermore, these findings helped us to evaluate our system in a relevant industrial context.

Our first result could proof, that there is absolutely no product conversion if ATP is missing in the reaction. As stated in the introduction this was an expected result.

Additionally, the necessity of a system which regenerates ATP was proven, by performing the reaction in absence of such a system. If there is no ATP in excess, the reaction comes to a stop fairly quickly after the given ATP is used up completely. Nevertheless, a little amount of conversion could be observed.

While monitoring the reaction with the usually used cofactor regeneration system we could observe a significant amount of product conversion. The kinases transfer the phosphate groups of the polyphosphate in the reaction vessel continuously to regenerate ATP from ADP. This keeps the reaction going.

The whole cell approach showed a similar product conversion to the in vitro approach. It has to be considered, that this approach has a way higher enzyme concentration, than the in vitro approach. Additionally, you can observe the formation of an unwanted side product, which has a negative effect on the yield.

![](https://static.igem.org/mediawiki/2020/2/26/T--Aachen--PC2.png)

![](https://static.igem.org/mediawiki/2020/1/1c/T--Aachen--PC3.png)

![](https://static.igem.org/mediawiki/2020/b/b3/T--Aachen--PC1.png)

![](https://static.igem.org/mediawiki/2020/1/15/T--Aachen--PC4.png)

### References

**[1] One-Pot Assembly of Complex Giant Unilamellar Vesicle-Based Synthetic Cell, Kerstin Göpfrich, Barbara Haller, Oskar Staufer, Yannik Dreher, Ulrike Mersdorf, Ilia Platzman, and Joachim P.
Spatz, ACS Synthetic Biology 2019 8 (5), 937-947 DOI: 10.1021/acssynbio.9b00034**
**[2] Strohmeier GA, Eiteljörg IC, Schwarz A, Winkler M. Enzymatic One-Step Reduction of Carboxylates to Aldehydes with Cell-Free Regeneration of ATP and NADPH. Chemistry. 2019 Apr
26;25(24):6119-6123. doi: 10.1002/chem.201901147. Epub 2019 Apr 5. PMID: 30866114; PMCID: PMC6563805**
**[3] Ritzmann N, Thoma J, Hirschi S, Kalbermatter D, Fotiadis D, Müller DJ. Fusion Domains Guide the Oriented Insertion of Light-Driven Proton Pumps into Liposomes. Biophys J. 2017 Sep 19;113(6):1181-1186. doi: 10.1016/j.bpj.2017.06.022. Epub 2017 Jul 8. PMID: 28697898; PMCID: PMC5607040**
**[4] Runswick Michael J., Bason John V., Montgomery Martin G., Robinson Graham C., Fearnley Ian M. and Walker John E., 2013, The affinity purification and characterization of ATP synthase complexes from mitochondria, Open Biol.3120160http://doi.org/10.1098/rsob.120160**

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

# Team:Aachen/Results

iGEM Aachen - Results

# Project Results

### Success does not come instantly, it is gained drop by drop

During the competition we as a team worked on several very different sides of our project. We went even further, outside the laboratory, to create models and hardware for M.A.R.S. and took our project to the new levels by creating
meaningful discourse with experts, companies and people of every imaginable background, to communicate science.

In the end, all of our efforts and activities came together and created a complete picture. Here, we dissect all of the different sides of our project and present to you our main results on each of them.

*Use the Pipette by clicking on it to see our main project results.*

![mars gif](https://static.igem.org/mediawiki/2020/2/23/T--Aachen--mars.gif)

![](https://static.igem.org/mediawiki/2020/7/78/T--Aachen--pipette_voll.png)

![1](https://static.igem.org/mediawiki/2020/4/45/T--Aachen--drop.png)

Lab Results

* Successful liposome formation
* Successful identification of magnetic particle binding peptide
* Binding tests on liposomes with different candidate peptides
* Implementation testing in a real industrial context at FZ Jülich

[![](https://static.igem.org/mediawiki/2020/f/f2/T--Aachen--spaceship_right.png)](https://2020.igem.org/Team%3AAachen/Experiments#Results)

[Go to detailed results](https://2020.igem.org/Team%3AAachen/Experiments#Results)

![1](https://static.igem.org/mediawiki/2020/4/45/T--Aachen--drop.png)

HP & IHP

* Several very insightful discussions with experts on topics related to M.A.R.S.
* Detailed reflection of the impact and responsibilities of M.A.R.S. and us as a team
* Communication with people and companies outside of synthetic biology or science in general

[![](https://static.igem.org/mediawiki/2020/f/f2/T--Aachen--spaceship_right.png)](https://2020.igem.org/Team%3AAachen/Human_Practices)

[Go to detailed results](https://2020.igem.org/Team%3AAachen/Human_Practices)

![1](https://static.igem.org/mediawiki/2020/4/45/T--Aachen--drop.png)

Hardware

* Design of a mostly 3D-printable bioreactor
* Modeling of the complete bioreactor as well as single components
* Start of production of parts of the reactors shaking mechanism
* Coded the server side communication protocol for an IoT platform

[![](https://static.igem.org/mediawiki/2020/f/f2/T--Aachen--spaceship_right.png)](https://2020.igem.org/Team%3AAachen/Hardware)

[Go to detailed results](https://2020.igem.org/Team%3AAachen/Hardware)

![1](https://static.igem.org/mediawiki/2020/4/45/T--Aachen--drop.png)

Modeling

* Successful molecular dynamics simulation of all components of our system
* Extensive mathematical modelling
* Parameter optimizations for M.A.R.S. application at FZ Jülich
* Creation of visualizations of the molecular mechanisms of M.A.R.S.

[![](https://static.igem.org/mediawiki/2020/f/f2/T--Aachen--spaceship_right.png)](https://2020.igem.org/Team%3AAachen/Model)

[Go to detailed results](https://2020.igem.org/Team%3AAachen/Model)

![1](https://static.igem.org/mediawiki/2020/4/45/T--Aachen--drop.png)

Science Communication

* Creation of a network of different projects connected to our educational framework “Science for everyone”
* Intensive discussions about science, our project, the chances and responsibilities of synthetic biology
* Communication and exchange between people, from various backgrounds. Newcomers, prospective and established scientists, as well as the broad public

[![](https://static.igem.org/mediawiki/2020/f/f2/T--Aachen--spaceship_right.png)](https://2020.igem.org/Team%3AAachen/Education)

[Go to detailed results](https://2020.igem.org/Team%3AAachen/Education)