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Method 1

Protein Building Blocks

Protein building Blocks (without additional Streptavidin) . The single building blocks contain N- and C-terminal catcher-tag elements to form a permanent lock-key system. The polymer is build of two different building blocks as one single building block cannot be handled without instant polymerization. Mixing of the two building blocks start the polymerization via condensation reaction leading to a biopolymer tissue.

Pilis/Carbon Nano Tubes

Geobacter-conductive pili. The bacteria geobacter is a strictly anaerobic stem, which can produce electrically conductive proteins (Pili). Originally, they are used for their own energy gain however, they can also be used as conductive layer in our dielectric elastomer actuator and replace the carbon nanotubes as ecologically friendly alternative.

Carbon Nanotubes (CNTs) are tubular architectures made of pure carbon. Every single carbon atom is sp2 hybridized and arranged in a hexagonal pattern. One can distinguish between Single Walled Carbon Nanotubes (SWCNTs) and Multi Walled Carbon Nanotubes (MWCNTs). The fabrication of the tubes can either be achieved by laser ablation of graphite or with a catalyst in a carbon rich gas phase. As pure carbon material, they are light-weight and flexible and robust. Further their thermal and electric conductivity is highly remarkable.

Artificial Muscle

Dielectric Elastomer Actuators (DEAs) are made of alternating layers of elastic and conductive material e.g. CNTs forming a stacked capacitor. The top and bottom contacts are metallic electrodes. By application of a voltage the elastomer between the cathodes and anodes is compressed in z-dimension and expands in the x,y-plane. This leads to a muscle-like contraction of the whole system.

Method 2

Protein Building Blocks

Protein building Blocks (with additional Streptavidin). The single building blocks contain N- and C-terminal catcher-tag elements with a streptavidin in the center of the protein. The catcher-tag system binds a permanent lock-key system to each other. The streptavidin unit can bind to the biotin functionalized azo dye to integrate the molecules into the polymer tissue.

Azo Dye
 

Molecular Machines (Azo dyes) can fulfill motion on a molecular length scale. The contraction of the dye is driven by light, which changes the conformation of the -N=N- bond from trans to cis. Implemented in high number in a tissue they can lead to contraction of the whole tissue. The invention of such small motors was awarded the nobel prize in chemistry in 2016.

Artificial Muscle

Muscle tissue with molecular machines can be obtained by combining biopolymeric tissue with an integrated streptavidin moiety. The streptavidin can bind a biotin functionalized azo dye leading to a further cross-linking. Via light-irradiation the azo dye molecules change their conformation and contract the tissue. Thermal or irradiation with a longer wavelength restores the position of the azo dye and the tissue relaxes to the original state.

The work, which is and can be done by robots is continuously increasing as well as the number of steps where complex shaped matter has to be handled. For this purpose, soft robotics are essential to prevent damage from the material. Current materials for soft robotics are based on silicones and related polymeric materials. Combined with electrically conductive materials in alternating layers, they form dielectric elastomer actuators (DEAs), which serve as muscle in robot arms. Silicones and elastic polymers can be counted to rather cost-efficient materials. However, considering the production of the material a high amount of electricity and chemical effort must be applied. This can be circumvented by a production from E. coli, where the organism produces a economically friendly biopolymer with the desired properties. The biosynthesis of the polymer building blocks, safes a significant amount of resources and energy.

The fabrication of the silicones to a device is carried out under elevated temperatures, where high accuracy at a micrometer scale is a crucial factor. This accuracy must also be maintained for the device made of biopolymer, which can be realized with modern 3D printers. Further, the 3D-printer is operating under ambient conditions, which safes money in the fabrication process. Due to the material properties, a special issue is the degradation of the biopolymer in dependence of time. A degradation of the material can mainly be circumvented by using the cell-free peptides, which prevents the consumption of the peptides by the cells. However, oxygen and mechanic stress are main issues, which have to be tackled. Oxygen can be excluded by instant packing of the material, whereas the mechanic durability of this material is still unknown. However, a comparison with a hydrogel polyacrylate based or a disposable DEA implies a lifetime of 100 to 2960 cycles.[1,2]

Biopolymer based DEA Silicone based DEA
Raw materials (price) 6 € /g ink [3,4] Av. 0,10 € /g ink[5]
Fabrication 3D printing 3D aerosol jet printing @ 80°C[6]
Solvent Water Isopropanol/terpineol[6]
Weight est. 1 g/mL (density of water) 1.1-1.3 g/mL [7]
Waste Biodegradable polymer; Simple regain of conductive material Recycling of silicone possible, regain of conductive material complex to achieve
References [1] WALKER, Stephanie, et al. Using an environmentally benign and degradable elastomer in soft robotics. International Journal of Intelligent Robotics and Applications, 2017, S. 1-19. [2] https://arxiv.org/abs/1409.2611 [3] MACEWAN, Sarah R.; CHILKOTI, Ashutosh. Elastin‐like polypeptides: Biomedical applications of tunable biopolymers. Peptide Science, 2010, 94. Jg., Nr. 1, S. 60-77. [4] http://www.formedium.com/eu/products/escherichia-coli-media/media-for-optimal-cell-growth-and-yield-of-e-coli-cultures.html [5] MADSEN, Frederikke B., et al. The Current State of Silicone‐Based Dielectric Elastomer Transducers. Macromolecular rapid communications, 2016, 37. Jg., Nr. 5, S. 378-413. [6] REITELSHÖFER, Sebastian, et al. Aerosol-Jet-Printing silicone layers and electrodes for stacked dielectric elastomer actuators in one processing device. In: Electroactive Polymer Actuators and Devices (EAPAD) 2016. International Society for Optics and Photonics, 2016. S. 97981Y. [7] http://www.chemie.de/lexikon/Silikone.html

Timeline

14
Sep

Synthesis 1,3-bis(-(4-iodophenyl)diazenyl)benzene

Chemical equation:

The same reaction mixture as on 09/12/17 was purified via column chromatography (EtOH/DCM, 1:19) affording a yellowish-orange fraction 1, a red fraction 2 and a yellowish-orange fraction 3.

12
Sep

Synthesis 1,3-bis(-(4-iodophenyl)diazenyl)benzene

Chemical equation:

Procedure:

The reaction mixture from 09/07/2017, which was dissolved in Cyclohexane, turned orange and a black precipitate was formed. The mixture was filtered and the residue again was dissolved in 150 mL of Cyclohexane. The dried reaction mixture from 09/08/2017 purified via column chromatography (EtOH/DCM, 1:19) affording the following fractions:

Yield:

Description of the product
Fraction 1 Orange liquid
Fraction 2 Reddish-orange liquid
Fraction 3 Red liquid
Fraction 4 Violet liquid
Fraction 5 Deep red liquid
Fraction 6 Violet liquid
Fraction 7 Violet-bluish liquid

A thin-layer chromatography was performed with the fractions 1-6. Only in the TLC of fraction number 3 an orange dot was visible.

08
Sep

Synthesis 1,3-bis(-(4-iodophenyl)diazenyl)benzene

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
4-iodo-1- nitrosobenzene 233,00 - - 0,13 0,56
Phenylenediamine 108,14 - - 0,03 0,28
Acetic acid 60,05 1,05 3 - -

Procedure:

Phenylenediamine (0,03 g; 0,28 mmol) acid in 3 mL acetic acid was added to 4-iodo- 1-nitrosobenzene (0,13 g; 0,56mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure.

07
Sep

Synthesis 1,3-bis(-(4-iodophenyl)diazenyl)benzene

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
4-iodo-1- nitrosobenzene 233,00 - - 0,215 0,92
Phenylenediamine 108,14 - - 0,05 0,46
Acetic acid 60,05 1,05 2 - -

Procedure:

Phenylenediamine (0,05 g; 0,46 mmol) acid in 2 mL acetic acid was added to 4-iodo- 1-nitrosobenzene (0,215 g; 0,92mmol). The reaction mixture was stirred at room temperature over night. The reaction mixture was concentrated under reduced pressure and purified via column chromatography (EtOH/DCM, 1:19). Acetic Acid was removed using a vacuum oven. Following the product was dissolved in Cyclohexane.

29
Aug

Synthesis

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [mg] n [mmol]
Biotin azide 401,2 - - 0,255 0,63
CuSO4 249,69 - - 0,013 0,0016
Sodium ascorbate 176,13 - - 0,011 0,063
Fluorenol 206,24 - - 0,130 0,63
H2O 18,00 - 0,75 - -
Isopropyl alcohol 60,10 0,78 0,25 - -
DCM 84,93 1,33 4,00 - -

Procedure:

Quaterthiophene (0,745 g; 2,25 mmol) and N-bromosuccinimide (0,505 g; 2,84 mmol) were dissolved in a mixture of CHCl 3 (2,85 mL) and cold AcOH (2,85 mL). The reaction mixture then was shielded from light and stirred over night at room temperature. Following the solvents were evaporated.

29
Aug

Synthesis of (Quaterthiophene-5-ylethynyl)trimethylsilane

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
α-Bromoquaterhiophene 409,68 - - 0,4 0,98
TMSA - 0,67 0,167 0,112 -
Pd(PPhy3)2Cl2 701,90 - - 0,02 0,03
Cul 190,44 - - 0,01 0,05
Net3 - 0,73 0,4 - -
THF - 0,89 4,6 - -

Procedure:

The reaction mixture of α-Bromoquaterhiophene (0,4 g; 0,98 mmol), TMSA (0,167 mL), Pd(PPh3)2Cl2 (0,02 g; 0,03 mmol) and CuI (0,01 g; 0,03 mmol) in Net3 (0,4 mL) and THF (4,6 mL) was heated at 65°C over night under nitrogen atmosphere. The Solvent was removed by evaporation.

28
Aug

Synthesis of α-Bromquaterthiophene

Chemical equation:

Reagents:

M [g/mol] V [mL] m [mg] n [mmol]
Quaterhiophene 330,50 - - 0,745 2,25
N-bromosuccinimide 177,99 - - 0,505 2,84
CHCl3 119,38 1,48 2,85 - -
AcOH 60,05 1,05 2,85 - -

Procedure:

Quaterthiophene (0,745 g; 2,25 mmol) and N-bromosuccinimide (0,505 g; 2,84 mmol) were dissolved in a mixture of CHCl 3 (2,85 mL) and cold AcOH (2,85 mL). The reaction mixture then was shielded from light and stirred over night at room temperature. Following the solvents were evaporated.

28
Aug

Synthesis Biotin-azide ( II )

Chemical equation:

Reagents:

M [g/mol] V [mL] m [mg] n [mmol]
Azide 175,19 - - 1,08 6,1
Biotin 244,31 - - 1,47 6,0
DMAP 122,17 - - 0,58 4,7
DCC 206,33 - - 2,13 11,2
CH2Cl2 84,93 1,33 45 - -

Procedure:

A solution of 2-(2- (2-azidoethoxy)ethoxy)ethanol (1,08 g; 6,1 mmol) , Biotin (1,47 g; 6,0 mmol), DMAP ( 0,58 g; 4,7 mmol) and DCC (2,13 g; 11,2 mmol) in CH 2 Cl 2 (45 ml) was stirred at room temperature overnight under N 2 - atmosphere. The reaction mixture was filtered off and washed with a small volume of CH 2 Cl 2. Following the crude product was purified by silica gel chromatography column (CH 2 Cl 2 / MeOH, 30:1) and the solvent was removed in a rotary evaporator.

24
Aug

Synthesis Biotin-azide ( I )

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [mg] n [mmol]
Azide 175,19 - - 1,02 5,8
Biotin 244,31 - - 1,49 6,1
DMAP 122,17 - - 0,57 4,6
DCC 206,33 - - 2,11 10,2
CH2Cl2 84,93 1,33 45 - -

Procedure:

A solution of 2-(2- (2-azidoethoxy)ethoxy)ethanol (1,02 g; 5,8 mmol) , Biotin (1,49 g; 6,1 mmol), DMAP ( 0,57 g; 4,6 mmol) and DCC (2,11 g; 10,2 mmol) in CH 2 Cl 2 (45 ml) was stirred at room temperature overnight under N 2 - atmosphere. The reaction mixture was filtered off and washed with a small volume of CH 2 Cl 2. Following the crude product was purified by silica gel chromatography column (CH 2 Cl 2 / MeOH, 30:1) and the solvent was removed in a rotary evaporator.

Yield:

Product M [g/mol] m[mg] n[mmol]
Biotin-azide (crude product) 401,48 1,628 4,05
Biotin-azide (dried) 401,48 1,188 2,95

23
Aug

Synthesis 2-(2-(2-azidoethoxy)ethoxy)ethanol

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
2-(2-(2-chloroethoxy)ethoxy)ethano l 168,62 1,16 2,16 2,51 14,9
NaN3 65,01 1,85 - 1,00 15,4
DMF 73,10 0,95 25 - -
THF 72,11 0,89 25 - -

Procedure:

At first 2-(2- (2-chloroethoxy)ethoxy)ethanol (2,51 g; 14,9 mmol) and NaN 3 (1,00 g; 15,4 mmol) were reacted in DMF (25 mL) overnight at 100°C. The mixture following cooling down was diluted with THF (25 mL) and filtered. After the solvent was removed, the residue was dissolved in diethyl ether (100 mL), washed with brine (20 mL) and dried over MgSO 4 . 2-(2- (2-azidoethoxy)ethoxy)ethanol was isolated as a yellowish orange solid.

Yield:

Product M [g/mol] m[mg] n[mmol]
2-(2-(2-azidoethoxy)ethoxy)ethanol 175,19 3,52 20,1
Description of product Yellowish orange solid

22
Aug

Synthesis 4-Iodo-1-Nitrosobenzen

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm 3 ] V [mL] m [mg] n [mmol]
4-iodo-1- nitrobenzene 249,01 - - 2,00 8,03
Zn 65,38 - - 1,39 21,26
NH4Cl 53,49 - - 0,7 13,09
FeCl3 × 6 H2O 270,29 - - 6,55 24,23
2-methoxyethanol 76,09 0,97 60 - -
H2O/EtOH (5:1) 18,00/46,07 1,00/0,79 72 - -

Procedure:

4-Iodo-1-nitrobenzen (2,00 g; 8,03 mmol) was dissolved in 60 mL 2-methoxyethanol. Zn dust (1,39 g; 21,26 mmol) and NH4Cl (0,7 g; 13,09 mmol) was added and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC (5:1 hexanes/EtOAc). After 45 minutes the solution was cooled to 0°C. FeCl3 × 6 H2O (6,55 g; 24,23 mmol) was solved in 72 mL H2O/EtOH (5:1) and added to the reaction mixture which then was stirred 3 hours at 0°C. Following the reaction mixture was extracted with EtOAc (3 × 80 mL), washed with brine and dried over MgSO4. The received solution was concentrated under reduced pressure and purified by silica gel column chromatography (hexanes/EtOAc, 50:1). The solvent was removed and the product 4-iodo-1-nitrosobenzen (0,472 g; 2,03 mmol) was received as a green solid.

Yield:

Product M [g/mol] m[mg] n[mmol]
4-iodo-1-nitrosobenzen (fraction 5 and 6) 233,0 0,472 2,03
Description of product Green solid

16
Jun

Synthesis 3,5-bis((4-iodophenyl)diazenyl)benzoic acid

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
4-iodo-1-nitrosobenzene 233,00 - - 0,055 0,24
Acetic acid 152,20 - - 0,017 0,11
Acetic acid 60,05 1,05 2 - -

Procedure:

3,5-diaminobenzoic (0,017 g; 0,11 mmol) acid in 2 mL acetic acid was added to 4- iodo-1- nitrosobenzene (0,055 g; 0,24 mmol). The reaction mixture was stirred at room temperature for four days. The reaction mixture was concentrated under reduced pressure and purified via column chromatography (EtOH/DCM, 1:19).

16
Jun

Synthesis 3,5-bis(4-(trimethylsilyl)phenyl)diazenyl)benzoic acid

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
4-iodo-1-nitrosobenzene 233,00 - - 0,125 0,53
Acetic acid 152,20 - - 0,048 0,32
Acetic acid 60,05 1,05 4 - -

(Fraction 3 and 4 of trimethyl((4-nitrosophenyl)ethynyl)silane from May 31th were added together)

Procedure:

3,5-diaminobenzoic (0,048 g; 0,32 mmol) acid in 4 mL acetic acid was added to trimethyl((4-nitrosophenyl)ethynyl)silane (0,125 g; 0,53 mmol). The reaction mixture was stirred at room temperature for four days. The reaction mixture was concentrated under reduced pressure and purified via column chromatography (EtOH/DCM, 1:19).

08
Jun

Synthesis 4-Iodo-1-Nitrosobenzen

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
4-iodo-1-nitrosobenzene 249,01 - - 4,03 16,2
Zn 65,38 - - 2,37 36,2
NH4Cl 53,49 - - 1,42 26,5
FeCl3 × 6 H2O 270,29 - - 13,09 48,4
2-methoxyethanol 76,09 0,97 130 - -
H2O/EtOH (5:1) 18,00/46,07 1,00/0,79 144 - -

Procedure:

4-Iodo-1-nitrobenzen (4,05 g; 16,3 mmol) was dissolved in 130 mL 2-methoxyethanol. Zn dust (2,37 g; 36,2 mmol) and NH4Cl (1,41 g; 26,4 mmol) was added and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC (5:1 hexanes/EtOAc). After 30 minutes the solution was cooled to 0°C. FeCl3 × 6 H2O (13,00 g; 48,1 mmol) was solved in 144 mL H2O/EtOH (5:1) and added to the reaction mixture which then was stirred 3 hours at 0°C. Following the reaction mixture was extracted with EtOAc, washed with brine and dried over MgSO4. The yielded solution was concentrated under reduced pressure and purified by silica gel column chromatography (hexanes/EtOAc, 50:1). The solvent was removed and the product 4-iodo-1-nitrosobenzen (0,76 g; 3,3 mmol; 20%) was received as a green solid.

Yield:

Product 4-iodo-1-nitrosobenzen
M [g/mol] m[mg] n[mmol] Description of Product
Fraction 1 233,0 1,147 5,0 Green Solid
Fraction 1 233,0 0,098 0,4 Green Solid

31
May

Synthesis trimethyl((4-nitrosophenyl)ethynyl)silane

Chemical equation:

Reagents:

M[g/mol] V[mL] m[mg] n[mmol]
Cul 190,45 - 0,057 0,3
Pd(PPh3)2Cl2 701,90 - 0,105 0,2
TMS 98,22 0,55 - -
TEA 101,19 0,55 - -
4-iodo-1-
nitrosobenzen		 232,92 - 1,17 5,0
THF 72,11 10,0 - -

Procedure:

4-iodo-1-nitrosobenzen (1,17 g, 5,0 mmol), CuI (0,057 g, 0,3 mmol) and Pd(PPh3)2Cl2 (0,105 g, 0,2 mmol) in TMS (0,55 mL), TEA (0,55 mL) and THF (10,0 mL) were stirred at 55°C under nitrogen overnight. The reaction mixture was washed with brine, concentrated under reduced pressure and purified by silica gel column chromatography (hexanes/EtOAc, 35:1).

Yield:

Product trimethyl((4-nitrosophenyl)ethynyl)silane
M [g/mol] m[mg] n[mmol] Description of product
Fraction 2 203,32 0,090 0,44 Orange solid
Fraction 3 203,32 0,094 0,46 Yellow solid
Fraction 4 203,32 0,031 0,15 Brown solid

23
May

Synthesis 3,5-bis((4-((trimethylsilyl)ethynyl)phenyl)
diazenyl)benzoic acid

Chemical equation:

Reagents:

M [g/mol] V [mL] m [mg] n [mmol]
Cul 190,45 - 14,80 0,072
Pd(PPh3)2Cl2 701,90 - 25,59 0,036
TMS 98,22 0,2 - 1,44
TEA 101,19 0,2 - 1,44
3,5-bis((4- iodophenyl)diazenyl)-benzoic acid 328,19 - 251,51 1,2
THF 72,11 5,0 - -

Procedure:

3,5-bis((4-iodophenyl)diazenyl)-benzoic acid (251,51 mg, 1,2 mmol), CuI (14,80 mg, 0,072 mmol) and Pd(PPh3)2Cl2 (25,59 mg, 0,036 mmol) in TMS (0,2 mL), TEA (0,2 mL) and THF (5,0 mL) were stirred at 55°C under nitrogen overnight. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (EtOH/DCM, 1:19).

Yield:

Product M [g/mol] m[mg] n[mmol]
3,5-bis((4-((trimethylsilyl)-
ethynyl)phenyl)diazenyl)-y
benzoic acid		 522,76 0,218 0,4
Yield Practically: 33%
Description of product Orange solid

16
May

Synthesis 4-Iodo-1-Nitrosobenzen

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
4-iodo-1-nitrosobenzene 249,01 - - 4,05 16,3
Zn 65,38 - - 2,37 36,2
NH4Cl 53,49 - - 1,41 26,4
FeCl3 × 6 H2O 270,29 - - 13,00 48,1
2-methoxyethanol 76,09 0,97 130 - -
H2O/EtOH (5:1) 18,00/46,07 1,00/0,79 144 - -

Procedure:

4-Iodo-1-nitrobenzen (4,05 g; 16,3 mmol) was dissolved in 130 mL 2-methoxyethanol. Zn dust (2,37 g; 36,2 mmol) and NH4Cl (1,41 g; 26,4 mmol) was added and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC (5:1 hexanes/EtOAc). After 30 minutes the solution was cooled to 0°C. FeCl3 × 6 H2O (13,00 g; 48,1 mmol) was solved in 144 mL H2O/EtOH (5:1) and added to the reaction mixture which then was stirred 3 hours at 0°C. Following the reaction mixture was extracted with EtOAc, washed with brine and dried over MgSO4. The yielded solution was concentrated under reduced pressure and purified by silica gel column chromatography (hexanes/EtOAc, 50:1). The solvent was removed and the product 4-iodo-1-nitrosobenzen (0,76 g; 3,3 mmol; 20%) was received as a green solid.

Yield:

Product M [g/mol] m[mg] n[mmol]
4-iodo-1-nitrosobenzen 233,0 0,76 3,3
Yield Literature: 82% Practically: 20%
Description of product Green solid

06
Apr

Synthesis 4-Iodo-1-Nitrosobenzen

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
4-iodo-1-nitrosobenzene 249,01 - - 3,991 16,03
Zn 65,38 - - 2,388 36,52
NH4Cl 53,49 - - 1,386 25,91
FeCl3 × 6 H2O 270,29 - - 13,108 48,49
2-methoxyethanol 76,09 0,97 120 - -
H2O/EtOH (5:1) 18,00/46,07 1,00/0,79 144 - -

Procedure:

4-Iodo-1-nitrobenzen (3,991 g; 16,03 mmol) was dissolved in 120 mL 2-methoxyethanol. Zn dust (2,388 g; 36,52 mmol) and NH4Cl (1,386 g; 25,91 mmol) was added and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC (5:1 hexanes/EtOAc). After 30 minutes the solution was cooled to 0°C. FeCl3 × 6 H2O (13,108 g; 48,49 mmol) was solved in 144 mL H2O/EtOH (5:1) and added to the reaction mixture which then was stirred 3 hours at 0°C. Following the reaction mixture was extracted with EtOAc, washed with brine and dried over MgSO4. The yielded solution was concentrated under reduced pressure and purified by silica gel column chromatography (hexanes/EtOAc, 50:1). The solvent was removed and the product 4-iodo-1-nitrosobenzen (0,541 g; 2,3 mmol; 14%) was received as a green solid.

Yield:

Product M [g/mol] m[mg] n[mmol]
4-iodo-1-nitrosobenzen 233,0 0,541 2,3
Yield Literature: 82% Practically: 14%
Description of product Green solid

06
Apr

Synthesis 3,5-bis((4-iodophenyl)diazenyl)benzoic acid

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
4-iodo-1-nitrosobenzene 233,00 - - 1,2 5,15
Acetic acid 152,20 - - 0,32 2,6
Acetic acid 60,05 1,05 30 - -

Procedure:

3,5-diaminobenzoic acid (0,32 g; 2,60 mmol) and 30 mL acetic acid was added to 4-iodo-1- nitrosobenzene (1,2 g; 5,15 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure and purified via column chromatography (EtOH/DCM, 1:19).

Yield:

Product M [g/mol] m[mg] n[mmol]
3,5-bis((4-iodophenyl)
diazenyl)benzoic
acid		 328,19 0,509 1,6
Description of product Orange/brown solid

05
Apr

Synthesis 4-Iodo-1-Nitrosobenzen

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
4-iodo-1-nitrosobenzene 249,01 - - 4,06 16,3
Zn 65,38 - - 2,53 35,7
NH4Cl 53,49 - - 1,43 26,7
FeCl3 × 6 H2O 270,29 - - 12,98 48,0
2-methoxyethanol 76,09 0,97 200 - -
H2O/EtOH (5:1) 18,00/46,07 1,00/0,79 144 - -

Procedure:

4-Iodo-1-nitrobenzene (4,06 g; 16,3 mmol) was dissolved in 200 mL 2-methoxyethanol. Zn dust (2,53 g; 35,7 mmol) and NH4Cl (1,43 g; 26,7 mmol) was added and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC (5:1 hexanes/EtOAc). After 30 minutes the solution was cooled to 0°C. FeCl3 × 6 H2O (12,98 g; 48,0 mmol) was solved in 72 mL H2O/EtOH (5:1) and added to the reaction mixture which then was stirred 3 hours at 0°C. Following the reaction mixture was extracted with EtOAc, washed with brine and dried over MgSO4. The yielded solution was concentrated under reduced pressure and purified by silica gel column chromatography (hexanes/EtOAc, 50:1). The solvent was removed and the product 4-iodo-1-nitrosobenzene (1,2 g; 5,2 mmol; 32%) was received as a green solid.

Yield:

Product M [g/mol] m[mg] n[mmol]
4-iodo-1-nitrosobenzen 233,0 1,2 5,2
Yield Literature: 82% Practically: 32%
Description of product Green solid

16
Mar

Synthesis 3,5-bis((4-iodophenyl)diazenyl)benzoic acid

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
4-iodo-1-nitrosobenzene 233,00 - - 0,863 3,7
Acetic acid 152,20 - - 0,299 2,0
Acetic acid 60,05 1,05 25 - -

Procedure:

3,5-diaminobenzoic (0,299 g; 2,0 mmol) acid in 25 mL acetic acid was added to 4-iodo-1- nitrosobenzene (0,863 g; 3,7 mmol). The reaction mixture was stirred at room temperature for four days. The reaction mixture was concentrated under reduced pressure and purified via column chromatography (EtOH/DCM, 1:19).
Fraction 3 was solved in 10 mL THF/water (1:1).
Fraction 4 was solved in 5 mL THF/water (1:1).

Yield:

Product M [g/mol] m[mg] n[mmol]
3,5-bis((4-iodophenyl)
diazenyl)benzoic
acid
Fraction 3 328,19 0,123 0,4
Fraction 4 328,19 0,026 0,08
Description of product Orange/brown solid

NMR

Azofarbstoff Fraktion 3 1. Synthese
NMR-Name: iGEM1

Azofarbstoff Fraktion(9)MeOH 1. Synthese
NMR-Name: iGEM2

14
Mar

Synthesis 4-Iodo-1-Nitrosobenzen

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
4-iodo-1-nitrosobenzene 249,01 - - 2,19 8,8
Zn 65,38 - - 1,46 22,3
NH4Cl 53,49 - - 0,71 13,3
FeCl3 × 6 H2O 270,29 - - 6,89 25,5
2-methoxyethanol 76,09 0,97 60 - -
H2O/EtOH (5:1) 18,00/46,07 1,00/0,79 72 - -

Procedure:

4-Iodo- 1-nitrobenzen (2,19 g; 8,8 mmol) was dissolved in 60 mL 2-methoxyethanol. Zn dust (1,46 g; 22,3 mmol) and NH 4 Cl (0,71 g; 13,3 mmol) was added and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC (5:1 hexanes/EtOAc). After 50 minutes the solution was cooled to 0°C. FeCl 3 × 6 H 2 O (6,89 g; 25,5 mmol) was solved in 72 mL H 2 O/EtOH (5:1) and added to the reaction mixture which then was stirred 3 hours at 0°C. Following the reaction mixture was extracted with EtOAc (3 × 80 mL), washed with brine and dried over MgSO 4 . The received solution was concentrated under reduced pressure and purified by silica gel column chromatography (hexanes/EtOAc, 50:1). The solvent was removed and the product 4-iodo- 1-nitrosobenzen (0,6 g; 2,6 mmol; 30%) was received as a green solid.

Yield:

Product M [g/mol] m[mg] n[mmol]
4-iodo-1-nitrosobenzen 233,0 0,6 2,6
Yield Literature: 82% Practically: 30%
Description of product Green solid

14
Mar

Synthesis 3,5-bis((4-iodophenyl)diazenyl)benzoic acid

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
4-iodo-1-nitrosobenzene 233,00 - - 0,60 2,6
Acetic acid 152,20 - - 0,18 1,2
Acetic acid 60,05 1,05 25 - -

Procedure:

3,5-diaminobenzoic (0,18 g; 1,2 mmol) acid in 25 mL acetic acid was added to 4-iodo- 1- nitrosobenzene (0,60 g; 2,6 mmol). The reaction mixture was stirred at room temperature for two nights. Following it was concentrated under reduced pressure.
¼ of the reaction mixture was purified via column chromatography (EtOH/DCM, 1:19).
¾ of the reaction mixture was washed with 5M NaOH and EtOAc. The aqueous phase was acidifies with AcOH to pH 2.
Observation: Green solid precipitates and the organic phase was orange. It was washed again with EtOAc and the combined extracts were concentrated under reduced pressure.

13
Mar

Synthesis 4-Iodo-1-Nitrosobenzen

Chemical equation:

Reagents:

M [g/mol] ρ [g/cm3] V [mL] m [g] n [mmol]
4-iodo-1-nitrosobenzene 249,01 - - 2,04 8,2
Zn 65,38 - - 1,24 19,0
NH4Cl 53,49 - - 0,7 13,1
FeCl3 × 6 H2O 270,29 - - 6,58 24,3
2-methoxyethanol 76,09 0,97 60 - -
H2O/EtOH (5:1) 18,00/46,07 1,00/0,79 72 - -

Procedure:

4-Iodo- 1-nitrobenzen (2,04 g; 8,2 mmol) was dissolved in 60 mL 2-methoxyethanol. Zn dust (1,24 g; 19,0 mmol) and NH 4 Cl (0,7 g; 13,1 mmol) was added and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC (5:1 hexanes/EtOAc). After 45 minutes the solution was cooled to 0°C. FeCl 3 × 6 H 2 O (6,58 g; 24,3 mmol) was solved in 72 mL H 2 O/EtOH (5:1) and added to the reaction mixture which then was stirred 3 hours at 0°C. Following the reaction mixture was extracted with EtOAc (3 × 80 mL), washed with brine and dried over MgSO 4 . The received solution was concentrated under reduced pressure and purified by silica gel column chromatography (hexanes/EtOAc, 50:1). The solvent was removed and the product 4-iodo- 1-nitrosobenzen (0,863 g; 3,7 mmol; 45%) was received as a green solid.

Yield:

Product M [g/mol] m[mg] n[mmol]
4-iodo-1-nitrosobenzen 233,0 0,863 3,7
Yield Literature: 82% Practically: 45%
Description of product Green solid
Show Timeline

Materials


Plasmids

  1. pBAD/Myc-His
  2. pSB1C3
Inserts/ constructs for pBAD/ Myc-His plasmid
  1. a) 3-Spytag-ELP5-HisTag (to be cut with NcoI and XhoI)
  2. a) Spytag-Streptavidin-ELP5-SnoopCatcher (to be cut with XhoI and HindIII-HF)
  3. a) HisTag-Snooptag-ELP5-Spycatcher (to be cut with XhoI and HindIII-HF)
  4. a) Kationisch kurz (to be cut with NcoI and XhoI)
  5. a) Anionisch kurz (to be cut with NcoI and XhoI)
  6. a) W51W54 (to be cut with HindIII-HF and BssHII)
Inserts/ constructs for pSB1C3 plasmid
  1. b) 3-Spytag-ELP5-HisTag (to be cut with EcoRI and PstI)
  2. b) Spytag-Streptavidin-ELP5-SnoopCatcher (to be cut with EcoRI and PstI)
  3. b) HisTag-Snooptag-ELP5-Spycatcher (to be cut with EcoRI and PstI)
  4. b) Kationisch kurz (to be cut with EcoRI and PstI)
  5. b) Anionisch kurz (to be cut with EcoRI and PstI)
  6. b) W51W54 (to be cut with EcoRI and PstI)

Timeline

01
Nov

Hydrogel with 12% gelatine, 0.1% alginate, 0.01 % salt and 87.89% azo-dye (AY38) solved water were tested.

After two hours irradiation of UV-Light the Hydrogel showed a fluctuation of 4mm.

30
Oct

Two preparations for each system (DEA and molecular machines) with

  1. 10 % gelatine
    0,1 % alginate
    0,01 % salt
    89,89 % water
  2. 12 % gelatine
    0,1 % alginate
    0,01 % salt
    87,89% water

The DEA System showed a deformation at 45 V and 28 mA.

27
Oct

Testing different concentrations of the hydrogel

  1. 10 % gelatine
    0,1 % alginate
    0,01 % salt
    89,89 % water
  2. 12 % gelatine
    0,1 % alginate
    0,01 % salt
    87,89% water
  3. 8 % gelatine
    0,1 % alginate
    0,01 % salt
    91,89% water
  4. 12 % gelatine
    0,1 % alginate
    0,01 % salt
    89,78% water

Sample b) had the best properties (elastic and stable).
The concentrations of sample b) were used for the following preparations:

  1. For testing the DEA system with nanotubes.

    A resistance of 110 mΩ was measured. This shows, that this system could work.


  2. For testing the system with molecular machines (solved azo dye instead of water).

    The hydrogel was irradiated with UV-light, but no deformation of the gel could be measured.


  3. For testing the TaqCFP (one with 100 µl TaqCFP in 3ml hydrogel and one with 120 µl TaqCFP in 3 ml hydrogel).

26
Oct

Isolation of the transformed plasmids from test tubes from 24.10. with PROMEGA PureYield plasmid minipreperation kit.

Restriction digest of transformed plasmids:
- 38.5 µl Transformed plasmid
- 4.5 µl 3.1 buffer
- 0.5 µl EcoIII
- 0.5 µl PstI
- 1 µl H2O
All samples were incubated at 37 °C for 20 h.
Gel run with all the transformed samples 1b-6b and Tag-CFP after the restriction digest:

4Sample 1b)_IV and 2b)_I were sent to the iGEM registry, since their bands were higher than the plasmid control.

Sample 6b)_III was sent to the iGEM registry, since its band was higher than the plasmid control.

Sample Tag-CFP1b)_IV was sent to the iGEM registry, since its insert band could be seen in the gel.

Production of hydrogel containing alginate, salt (CaCl2), gelatine and water. Testing three samples with different concentrations:

  1. 3 % gelatine
    0,1 % alginate
    0,01 % salt
    96,89 % water
  2. 5 % gelatine
    0,1 % alginate
    0,01 % salt
    94,89 % water
  3. 7,5 % gelatine
    0,1 % alginate
    0,01 % salt
    92,39 % water
  4. 10 % gelatine
    0,1 % alginate
    0,01 % salt
    89,89 % water

Sample d) had the best properties (elastic and stable).

24
Oct

Inoculation of 4 test tubes (4 ml LB-medium) per sample, each with one single colonies from transformation from 23.10.

23
Oct

Transformation of E.coli DH5α MCR with ligations of 20.10 with all samples 1b-6b and Tag-CFP.

20
Oct

PCR with purified Tag-CFP from 19.10.:

- 20 µl Tag-CFP
- 10 µl 5x Phusion DNA Polymerase Buffer
- 4 µl dNTPs (2,5 mM)
- 1.5 µl DMSO
- 1 µl Primer 1 (100 pmol/µl)
- 1 µl Primer 2 (100 pmol/µl)
- 0.5 µl Phusion DNA Polymerase
- 13 µl H2O

PCR protocol:
- Initial denaturation: 3 min, 96 °C
- Denaturation: 30 s, 96 °C
- Annealing: 30 s, 55 °C
- Elongation: 2 min, 68 °C
- Terminal elongation: 10 min, 68 °C
- Idle: 4 °C

Restriction digest of PCR-product of Tag-CFP:
- 2 µl Tag-CFP
- 1 µl 3.1 buffer
- 0.5 µl EcoIII
- 0.5 µl PstI
- 6 µl H2O
Restriction digest of samples 1b-6b (see 14.10.)
Restriction digest of pSB1C3 plasmid:
- 1 µl Plasmid
- 1 µl 3.1 buffer
- 0.5 µl EcoIII
- 0.5 µl PstI
- 0.5 µl DpnI
- 6.5 µl H2O
All samples were incubated at 37 °C for 1 h.
Ligation of pSB1C3 of both Tag-CFP and samples 1b-6b after restriction digest:
- 10 µl Plasmid
- 10 µl Insert
- 3 µl 10x T4 DNA Ligase Buffer
- 1 µl T4 DNA Ligase
- 6 µl H2O
All samples were incubated at room temperature over night.

19
Oct

Restriction digest of Tag-CFP insert in two equal samples:

- 0.5 µl Insert
- 1 µl Cutsmart buffer
- 1 µl BamHI
- 1 µl SalI
- 6.5 µl H2O
The samples was incubated at 37 °C for 20 min. Then it was dephosphorylated:
- 2 µl 10x rapid alkaline phosphatase reaction buffer
- 0.5 µl alkanline phosphatase
- 7.5 µl H2O
The samples was incubated at 37 °C for 1 h.
Gel run from the restriction digests of Tag-CFP was achieved.

Both Tag-CFP samples were extracted from the gel and purified via PROMEGA Gel Purification Kit.

18
Oct

Isolation of the transformed plasmids 1a-6a and 1b-6b and pBAD/Myc-His plasmid from test tubes from 16.10. with PROMEGA PureYield plasmid minipreperation kit. Then gel run from the isolated transformed plasmids.

Gel bands of 1a)_II, 1a)_IV, 2a)_I and 2a)_II were further used.

Gel bands of 3a)_I, 3a)_II, 4a)_I and 4a)_III were further used.

Gel bands of 5a)_II, 5a)_III, 6a)_II and 6a)_III were further used.

Gel bands of 1b)_I, 1b)_II, 2b)_I and 2b)_III were further used.

Gel bands of 3b)_II, 3b)_IV, 4b)_I and 4b)_II were further used.

Gel bands of 5b)_II, 5b)_IV, 6b)_I and 6b)_II were further used.
All used further samples where then digested with the appropriate restriction enzymes:
- 15 µl Insert
- 2 µl Cutsmart/3.1 buffer
- 0.5 µl NcoI/XhoI/HindIII-HF/EcoIII
- 0.5 µl XhoI/HindIII-HF/BssHII/PstI
- 2 µl H2O
All samples were incubated at 37 °C for 105 min.
A gel run was then achieved but in any sample no band could be seen or had the wrong size.

17
Oct

Gel run of transformed plasmids from 16.10.

Bands of 6a)_III and 6a)_II looked promising that the transformation was successful.
Inoculation of 4 test tubes (4 ml LB-medium) per sample, each with one single colonies from transformation from 16.10. and incubation at 37 °C over night.

16
Oct

Isolation of the transformed plasmids 3a)_I, 3a)_II, 5a)_I, 5a)_II, 6a)_I and 6a)_II and pBAD/Myc-His plasmid from test tubes from 10.10. with PROMEGA PureYield plasmid minipreperation kit.

Restriction digest with transformed plasmids:
- 20 µl Transformed plasmid
- 4 µl Cutsmart buffer
- 1 µl NcoI/XhoI/HindIII-HF
- 1 µl XhoI/HindIII-HF/BssHII

All samples were incubated at 37 °C for 1 h. except form samples 6a which were first incubated at 37 °C for 45 min with HindIII-HF and then heated to 50 °C for further 45 min after adding BssHII.
All samples were dephosphorylated with alkaline phosphatase by adding:
- 4.5 µl 10x rapid alkaline phosphatase reaction buffer
- 0.5 µl alkanline phosphatase
All samples were incubated at 37 °C for 1 h and then heat inactivated at 80 °C for 20 min.

Transformation of E. coli DH5α MCR ligations from 14.10. (for protocol see 18.07.).

14
Oct

3 restriction digests with pBAD/Myc-His plasmid


  1. - 30 µl Plasmid
    - 9 µl Cutsmart Buffer
    - 2 µl NcoI
    - 2 µl XhoI
    - 47 µl H2O
  2. - 20 µl Plasmid
    - 6 µl Cutsmart Buffer
    - 1.5 µl XhoI
    - 1.5 µl HindIII-HF
    - 31 µl H2O
  3. - 10 µl Plasmid
    - 3 µl Cutsmart Buffer
    - 1 µl HindIII-HF
    - 1 µl BssHII
    - 15 µl H2O

    4. Restriction digest with pSB1C3 plasmid:
    - 10 µl Plasmid
    - 5 µl 3.1 Buffer
    - 0.5 µl EcoIII
    - 0.5 µl PstI
    - 0.5 µl DpnI
    - 1.5 µl H2O

    Restriction digest with Inserts 1a-6a:
    - 2 µl Insert
    - 2 µl Cutsmart buffer
    - 0.5 µl NcoI/XhoI/HindIII-HF
    - 0.5 µl XhoI/HindIII-HF/BssHII
    - 15 µl H2O

    Restriction digest with Inserts 1b-6b:
    - 12 µl Insert
    - 2 µl 3.1 buffer
    - 0.5 µl EcoIII
    - 0.5 µl PstI
    - 5 µl H2O

    All samples were incubated at 37 °C for 1 h. The sample 3 was first incubated at 37 °C for 40 min and then heated to 50 °C for further 40 min.
    All samples were dephosphorylated with alkaline phosphatase by adding:

    1. - 10 µl 10x rapid alkaline phosphatase reaction buffer
      - 1.5 µl alkanline phosphatase
    2. - 7 µl 10x rapid alkaline phosphatase reaction buffer
      - 1 µl alkanline phosphatase
    3. for pBAD/Myc-His plasmid and for pSB1C3:
      - 3.5 µl 10x rapid alkaline phosphatase reaction buffer
      - 0.5 µl alkanline phosphatase
      - 1 µl H2O

    All samples were incubated at 37 °C for 60 min. After that all samples were heat inactivated at 80 °C for 20 min.

    Ligation with remains of digested inserts 1a-6a with pBAD/Myc-His plasmid:
    - 10 µl Plasmid
    - 20 µl Insert
    - 4 µl 10x T4 DNA Ligase Buffer
    - 1 µl T4 DNA Ligase
    - 5 µl H2O

    Ligation with remains of digested inserts 1b-6b with pBAD/Myc-His plasmid:
    - 2 µl Plasmid
    - 20 µl Insert
    - 3 µl 10x T4 DNA Ligase Buffer
    - 1 µl T4 DNA Ligase
    - 4 µl H2O

    All samples were incubated over night at room temperature.

13
Oct

PCR with all constructs 1a, 2a, 3a, 4a, 5a, and 6a as template DNA with associated primers (for protocol see 19.09).

PCR with pSB1C3 plasmid:
- 0.5 µl Template DNA
- 10 µl 5x Phusion DNA Polymerase Buffer
- 4 µl dNTPs (2,5 mM)
- 1.5 µl DMSO
- 1 µl SB-prep-3P-1 primer (100 pmol/µl)
- 1 µl SB-prep-2Ea primer (100 pmol/µl)
- 0.5 µl Phusion DNA Polymerase
- 30.5 µl H2O

The following PCR program was used for all PCRs:
- Initial denaturation: 3 min, 96 °C
- Denaturation: 30 s, 96 °C
- Annealing: 30 s, 55 °C
- Elongation: 2 min, 68 °C
- Terminal elongation: 10 min, 68 °C
- Idle: 4 °C

12
Oct

Isolation of the transformed plasmids from test tubes from 09.10. with PROMEGA PureYield plasmid minipreperation kit. A gel run could not be achieved due to the accidentally discarded transformed plasmids.

11
Oct

Restriction digest from samples 3a)_XI, 5a)_II, 5a)_VI, 5a)_VII and 5a)_VIII (from 04.10) and from samples 3a)_I and 3a)_II, 5a)_I, 5a)_II, 6a)_old_II and 6a)_old_III (from 09.10):

- 15 µl Insert
- 2 µl Cutsmart buffer
- 1 µl NcoI/XhoI/HindIII-HF
- 1 µl XhoI/HindIII-HF/BssHII
- 1µl H2O

All samples were incubated at 37 °C for 1 h. The samples containing 6a were first incubated at 37 °C for 40 min and then heated to 50 °C for further 40 min.
Gel run for all the samples after the restriction digest. Here, unfortunately the plasmid control was forgotten so the gels results could not have been evaluated.

10
Oct

Inoculation of 4 test tubes (4 ml LB-medium) per sample, each with one single colonies from transformation from 09.10.

09
Oct

Isolation of the transformed plasmids from test tubes from 07.10. with PROMEGA PureYield plasmid minipreperation kit followed by gel electrophoresis with 1 % agarose gel at 130 V..

The Gel bands of the samples 3a)_I and 3a)_II looked promising for a successful transformation. The other samples were all at the same height as the plasmid, so the ligation could not have been successful.

The Gel bands of the samples 5a)_I, 5a)_II, 6a)_old_II and 6a)_old_III (samples 6a)_old from 20.09) looked promising for a successful transformation. The other samples were all at the same height as the plasmid, so the ligation could not have been successful.
New transformation of E. coli DH5α MCR with plasmid (pBAD/Myc-His) (for protocol see 18.07.).

07
Oct

Inoculation of 4 test tubes (4 ml LB-medium) per sample, each with one single colony from the respective agar-plates (06.10.). The test tubes were incubated at 37 °C over night.

06
Oct

Inoculation of 4 test tubes (4 ml LB-medium) per sample, each with one single colony from the respective agar-plates (06.10.). The test tubes were incubated at 37 °C over night.

05
Oct

3 restriction digests with pBAD/Myc-His plasmid

  1. - 10 µl Plasmid
    - 5 µl Cutsmart Buffer
    - 1.5 µl NcoI/XhoI/HindIII-HF
    - 1.5 µl XhoI/HindIII-HF/BssHII
    - 32 µl H2O
  2. - 8 µl Plasmid
    - 4 µl Cutsmart Buffer
    - 1 µl NcoI/XhoI/HindIII-HF
    - 1 µl XhoI/HindIII-HF/BssHII
    - 25 µl H2O
  3. - 6 µl Plasmid
    - 3 µl Cutsmart Buffer
    - 1 µl NcoI/XhoI/HindIII-HF
    - 1 µl XhoI/HindIII-HF/BssHII
    - 19 µl H2O

All samples were incubated at 37 °C for 1 h. The sample 3 was first incubated at 37 °C for 40 min and then heated to 50 °C for further 40 min.
All samples were dephosphorylated with alkaline phosphatase by adding

  1. - 6 µl 10x rapid alkaline phosphatase reaction buffer
    - 1 µl alkanline phosphatase
    - 3 µl H2O
  2. - 4,5 µl 10x rapid alkaline phosphatase reaction buffer
    - 1 µl alkanline phosphatase
  3. - 3.5 µl 10x rapid alkaline phosphatase reaction buffer
    - 0.5 µl alkanline phosphatase
    - 1 µl H2O

All samples were incubated at 37 °C for 20 min. After that all samples were heat inactivated at 80 °C for 20 min.
Ligation with remains of digested inserts from 02.10.:
- 5 µl Plasmid
- 2 µl Insert
- 1 µl 10x T4 DNA Ligase Buffer
- 1 µl T4 DNA Ligase
- 1 µl H2O
All samples were incubated over night at room temperature.

04
Oct

Gel electrophoresis with 1 % agarose gel at 130 V of samples from ligation (02.10.). The gel bands from samples 1a-2a did not indicate any successful ligation

The Gel band of sample 3a)_XI was promising for a successful ligation. The other samples were all at the same height as the plasmid, so the ligation could not have been successful.

The Gel bands of the samples 5a)_II, 5a)_VI, 5a)_VII and 5a)_VIII were promising for a successful ligation. The other samples were all at the same height as the plasmid, so the ligation could not have been successful.
Plasmid isolation with PROMEGA PureYield plasmid miniprep kit with all test tubes from 02.10., followed by gel electrophoresis with 1 % agarose gel at 130 V. The plasmid bands were at the right size.

02
Oct

Inoculation of 8 test tubes (4 ml LB-medium) per each sample 1a-6a (from 20.09.) with a single colony and incubation for 7 h at 37 °C.

After that, the test tubes were stored at 4 °C. Gel electrophoresis with 1 % agarose gel at 130 V with the sample of the ligation of insert 6a from 29.09. Gel run displayed no bands for insert 6a. Therefore, the PCR of the sample was not successful.
New restriction digests for pBAD/Myc-His plasmid:

  1. - 30 µl pBAD/Myc-His plasmid
    - 9 µl Cutsmart Buffer
    - 3 µl NcoI
    - 3 µl XhoI
    - 45 µl H2O
  2. - 20 µl pBAD/Myc-His plasmid
    - 6 µl Cutsmart Buffer
    - 2 µl XhoI
    - 2 µl HindIII-HF
    - 30 µl H2O
  3. - 10 µl pBAD/Myc-His plasmid
    - 3 µl Cutsmart Buffer
    - 1 µl HindIII-HF
    - 1 µl BssHII
    - 15 µl H2O

All samples were incubated at 37 °C for 1 h, while sample 3 was first incubated at 37 °C for 40 min and then heated to 50 °C for further 40 min.

After that all samples were dephosphorylated with alkaline phosphatase by adding to the samples:

  1. - 10 µl 10x rapid alkaline phosphatase reaction buffer
    - 1 µl alkanline phosphatase
  2. - 7 µl 10x rapid alkaline phosphatase reaction buffer
    - 1 µl alkanline phosphatase
    - 3 µl H2O
  3. - 3.5 µl 10x rapid alkaline phosphatase reaction buffer
    - 0.5 µl alkanline phosphatase
    - 1 µl H2O

All samples were incubated at 37 °C for 20 min.
Ligation for all inserts 1a-6a:
- 5 µl pBAD/Myc-His plasmid
- 2 µl Insert
- 1 µl 10x T4 DNA Ligase Buffer
- 1 µl T4 DNA Ligase
- 1 µl H2O
All samples were incubated over night at room temperature.

29
Sep

Ligation of insert 6a with digested plasmid (pBAD/Myc-His, cut with HindIII-HF and BssHII):

- 10 µl pBAD/Myc-His plasmid
- 10 µl Insert
- 3 µl 10x T4 DNA Ligase Buffer
- 1 µl T4 DNA Ligase
- 6 µl H2O
The sample was incubated at 4 °C over the weekend.

New try to transfer Geobacter sulfurreducens in 150 ml medium in 300 ml Bottle (for protocol see 28.08. / 29.08.). The cells were transferred after 4 h of incubation of medium in the bag. The Geobacter were incubated at 28 °C for growth

4Sample 1b)_IV and 2b)_I were sent to the iGEM registry, since their bands were higher than the plasmid control.

Sample 6b)_III was sent to the iGEM registry, since its band was higher than the plasmid control.

Sample Tag-CFP1b)_IV was sent to the iGEM registry, since its insert band could be seen in the gel.

Production of hydrogel containing alginate, salt (CaCl2), gelatine and water. Testing three samples with different concentrations:

  1. 3 % gelatine
    0,1 % alginate
    0,01 % salt
    96,89 % water
  2. 5 % gelatine
    0,1 % alginate
    0,01 % salt
    94,89 % water
  3. 7,5 % gelatine
    0,1 % alginate
    0,01 % salt
    92,39 % water
  4. 10 % gelatine
    0,1 % alginate
    0,01 % salt
    89,89 % water

Sample d) had the best properties (elastic and stable).

28
Sep

Gel electrophoresis with samples (27.09.) on 1 % agarose gel at 130 V.

New PCR with insert 6 (for protocol see 19.09.)

The gel revealed that the samples may be right sized but because of the big sized band in the region of the restricted inserts the result could not be validated.

27
Sep

Gel electrophoresis with samples from 26.09 was repeated.

Samples 3a)_III, 4a)_I, 4a)_II, 5a)_I and 5a)_II looked promising and restriction digests were accomplished from these samples:
- 15 µl Transformed plasmid
- 2 µl Cutsmart buffer
- 1 µl NcoI/XhoI
- 1 µl XhoI/HindIII-HF
- 1 µl H2O
All samples were incubated at 37 °C for 1 h, heat inactivated for 20 min at 80 °C and stored at -20 °C.

26
Sep

Plasmid isolation with PROMEGA PureYield plasmid miniprep kit followed by gel electrophoresis with 1 % agarose gel at 130 V. Gel run failed, therefore new plasmid isolation was achieved. The transformed plasmid samples were stored at -20 °C over night.

25
Sep

Inoculation of test tubes with 4 ml of LB-medium with single colonies from the transformation (21.09.) and incubation at 37 °C over night.

21
Sep

Transformation of E. coli DH5α MCR with each ligation sample (for protocol see 18.07.). Agar-plates were stored at 4 °C.

20
Sep

Restriction digest with PCR products per sample (for all inserts 1a-6a with appropriate enzymes):

  1. - 48 µl Insert
    - 5 µl Cutsmart buffer
    - 1 µl NcoI/XhoI/HindIII-HF
    - 1 µl XhoI/HindIII-HF/BssHII
  2. - 2 µl pBAD/Myc-His plasmid
    - 1 µl Cutsmart Buffer
    - 1 µl NcoI/XhoI/HindIII-HF
    - 1 µl XhoI/HindIII-HF/BssHII
    - 5 µl H2O

All insert containing samples were incubated at 37 °C for 1 h. Samples containing plasmids were dephosphorylated with alkaline phosphatase by adding:
- 1.5 µl 10x rapid alkaline phosphatase reaction buffer
- 0.5 µl alkaline phosphatase
- 3 µl H2O
The plasmid containing samples were incubated at 37 °C for 20 min. After that ligations were started with the respective inserts and plasmids:
- 15 µl pBAD/Myc-His plasmid
- 55 µl Insert
- 8 µl 10x T4 DNA Ligase Buffer
- 1 µl T4 DNA Ligase
- 1 µl H2O
All samples were incubated over night at room temperature.

19
Sep

PCR with all constructs 1a, 2a, 3a, 4a, 5a, and 6a as template DNA with associated primers:

- 1 µl Template DNA
- 10 µl 5x Phusion DNA Polymerase Buffer
- 4 µl dNTPs (2,5 mM)
- 1.5 µl DMSO
- 2.5 µl forward primer (100 pmol/µl)
- 2.5 µl reverse primer (100 pmol/µl)
- 0.5 µl Phusion DNA Polymerase
The following PCR program was used:
- Initial denaturation: 3 min, 98 °C
- Denaturation: 20 s, 98 °C
- Annealing: 20 s, 56 °C
- Elongation: 50 s, 72 °C
- Terminal elongation: 7 min, 72 °C
- Idle: 4 °C

01
Sep

New restriction digest:

  1. - 3 µl pBAD/Myc-His plasmid
    - 4 µl Cutsmart Buffer
    - 1 µl NcoI
    - 1 µl XhoI
    - 31 µl H2O
  2. - 7 µl Insert 4a
    - 2 µl Cutsmart Buffer
    - 1 µl NcoI
    - 1 µl XhoI
    - 9 µl H2O
  3. - 6 µl insert 5a
    - 2 µl Cutsmart Buffer
    - 1 µl NcoI
    - 1 µl XhoI
    - 10 µl H2O

All insert containing samples were incubated at 37 °C for 1 h. The samples containing plasmids were dephosphorylated with alkaline phosphatase by adding:
- 4.5 µl 10x rapid alkaline phosphatase reaction buffer
- 0,5 µl alkanline phosphatase
The plasmid samples were then incubated at 37 °C for 20 min.
Ligation:
Two ligations were prepared, each with one of the inserts 4a or 5a
- 20 µl pBAD/Myc-His plasmid
- 20 µl Insert
- 5 µl 10x T4 DNA Ligase Buffer
- 1 µl T4 DNA Ligase
- 4 µl H2O
All samples were incubated over night at room temperature. The samples were discarded due to errors in constructs.

31
Aug

The plasmid minipreperation and the gel electrophoresis from 30.08 were repeated.

The gel displayed only wrong sized ligation fragments, since they run the same distance as the plasmid control.

30
Aug

Transformed plasmids from 29.08. were isolated with PROMEGA PureYield plasmid miniprep kit and a gel electrophoresis with 1 % agarose gel was run, but the control with the undigested plasmid was forgotten.

29
Aug

8 colonies were picked from each plate from 28.08. to inoculate test tubes and the plates were incubated over night at 37°C.


Geobacter transfer: - Bottle closed with septum cap in still sealed bag - Bag punctured with sterile syringe to draw in oxygen free gas - Septum of Geobacter containing test tube punctured with syringe to pull in Geobacter cells - Quick transfer to closed 1 L medium bottle trough septum - Incubated at 30°C for cell proliferation

28
Aug

The ligation products were transformed in E.coli DH5α MCR (for protocol see 18.07.)

Geobacter sulfurreducens and Geobacter metallireducens were transferred from sealed test tubes to 1 L media bottles:
- Open Medium bottle placed in 10 L sterile bag together with sterilized septum containing cap
- “Aerogen 2,5 L” and “Aerocult” packs activated and added to bag
- closed bag to be airtight
- Let stand over night

25
Aug

Restriction digest:


  1. - 2 µl pBAD/Myc-His plasmid - 3 µl Cutsmart Buffer - 1 µl NcoI - 1 µl XhoI - 23 µl H2O
  2. - 12 µl Insert 1a - 3 µl Cutsmart Buffer - 1 µl NcoI - 1 µl XhoI - 13 µl H2O
  3. - 2 µl pBAD/Myc-His plasmid - 3 µl Cutsmart Buffer - 1 µl XhoI - 1 µl HindIII-HF - 23 µl H2O
  4. - 19 µl Insert 2a - 3 µl Cutsmart Buffer - 1 µl XhoI - 1 µl HindIII-HF - 6 µl H2O

All insert containing samples were incubated for 1h at 37 °C. The plasmid containing samples were incubated for 45 min at 37 °C. The following was added afterwards: - 0,5 µl alkaline phosphatase - 3,5 µl 10x rapid buffer - 1 µl H2O The plasmid containing samples were then incubated for another 20 min at 37°C and then heat inactivated for 10 min at 80 °C. Hereafter ligation attempts were prepared:

  1. - 35 µl pBAD/Myc-His plasmid (cut with NcoI and XhoI) - 30 µl Insert 1a - 7 µl 10x T4 DNA Ligase Buffer - 1 µl T4 DNA Ligase
  2. - 35 µl pBAD/Myc-His plasmid (cut with XhoI and HindIII-HF) - 30 µl Insert 2a - 7 µl 10x T4 DNA Ligase Buffer - 1 µl T4 DNA Ligase

    3. All samples were incubated over night at room temperature.

18
Aug

Gel electrophoresis with undigested Inserts and plasmid. Gel run failed

17
Aug

Repeated restriction digest from 21.07. but following gel electrophoresis showed only bands of the control (see 08.08).

15
Aug

Restriction digest of isolated transformed plasmids:

- 10 µl transformed plasmid
- 2 µl Cutsmart Buffer
- 1 µl NcoI
- 1 µl XhoI
- 6 µl H2O
All samples were incubated for 1h at 37 °C. A gel electrophoresis with 1 % agarose gel was run with 6 µl 6x purple loading dye (NEB) added to each sample. Gel run failed.

14
Aug

The plasmids were isolated with PROMEGA PureYield plasmid miniprep kit and stored at -20 °C.

11
Aug

Four colonies from each sample were picked to inoculate a test tube with 4 ml of LB-medium. The samples were incubated over night at 37 °C.

10
Aug

The ligated samples were transformed (for protocol see 18.07.).

08
Aug

New cloning attempt like on 21.07. with added control sample containing:


- 10 µl pBAD/Myc-His plasmid
- 2 µl 10x T4 DNA ligase buffer
- 1 µl T4 DNA ligase
- 7 µl H2O
Medium for Geobacter growth was finished.

07
Aug

Further weighing in of salts for Geobacter medium.

04
Aug

Further weighing in of salts for Geobacter medium.

01
Aug

Gel electrophoresis was run with digested samples from 31.07. to check enzyme activity. Gel run failed.

31
Jul

New restriction digest (for protocol see 21.07.)

28
Jul

Weighing in of salts for medium for Geobacter sulfurreducens (for contents see DSMZ.de -> Geobacter sulfurreducens).

27
Jul

Repetition of the gel electrophoresis of the previous day but no change whatsoever.

26
Jul

The transformed plasmids from the 8 tubes were isolated with PROMEGA PureYield plasmid miniprep kit.

All samples were analysed via gel electrophoresis with 1 % agarose gel (stained with Midori green) for 45 min at 150 V. The gel displayed the bands of the transformed plasmids with inserts of 1a) and 2a) being at the same height as the pBAD/Myc-His plasmid. The transformation therefore did not work, since the expected bands needed to be higher in the gel due to their bigger size.

25
Jul

For each sample of the plated transformation from 25.07., four colonies were partly transferred on ¼ of a new ampicillin LB-plate. The rest of each colony was inoculated in a test tubes containing 4 ml of LB-medium. Both the test tubes and the plates were incubated over night at 37 °C.

24
Jul

Transformation of E. coli DH5α MCR with entire samples from ligation (for protocol see 18.07.).

21
Jul

Restriction digest:


  1. - 10 µl pBAD/Myc-His (~ 600 ng)
    - 3 µl Cutsmart Buffer
    - 1 µl NcoI
    - 1 µl XhoI
    - 15 µl H2O
  2. - 10 µl Insert 1a (100 ng)
    - 2 µl Cutsmart Buffer
    - 1 µl NcoI
    - 1 µl XhoI
    - 6 µl H2O
  3. - 10 µl Insert 2a (100 ng)
    - 2 µl Cutsmart Buffer
    - 1 µl NcoI
    - 1 µl XhoI
    - 6 µl H2O

All three samples were incubated at 37 °C for 1.5 h. After 1 h of incubation the pBAD/Myc-His plasmid containing sample was dephosphorylated with alkaline phosphatase by adding:
- 3.5 µl 10x rapid alkaline phosphatase reaction buffer
- 0.5 µl alkaline phosphatase
- 1 µl H2O
The sample was further incubated at 37 °C for 30 min. After an overall incubation time of 1.5 h, all contained enzymes were inactivated at 65 °C for 20 min followed by 2 min at 74 °C.
Ligation for each Insert 1a and 2a:
- 5 µl pBAD/Myc-His plasmid (100 ng)
- 20 µl Insert (100 ng)
- 4 µl 10x T4 DNA Ligase Buffer
- 1 µl T4 DNA Ligase
- 10 µl H2O
All samples were incubated over night at room temperature.

20
Jul

pBAD/Myc-His plasmids were isolated with PROMEGA PureYield plasmid miniprep kit and 15 Eppendorf tubes with ~ 65 ng/µl plasmid were obtained.

19
Jul

Three Erlenmeyer flasks (250 ml) were inoculated with each one colony from the agar plates and were shaken over night at 37°C.

18
Jul

Transformation of E. coli DH5α MCR with pBAD/Myc-His plasmid:

- 1 µl pBAD/Myc-His plasmid was added to 100 µl of frozen competent cells
- Incubation: on ice for 1 h
- Heat shock: 42 °C for 1 min
- 700 µl of preheated (37 °C) LB-medium was added
- Incubation: 37 °C for 1 h
- Centrifugation: 13000 x g for 2 min
- Liquid phase was discarded
- Cells were resuspended in remaining liquid (~ 100 µl)
- Entire samples were plated out on ampicillin containing LB-agar plates
- Incubation: over night at 37 °C

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The term “molecular machine” refers to a system that is able to perform mechanical movement on a nanoscopic scale by application of an external stimulus. In order to do so, such systems only consist of a small number of molecules. Depending on the nature of the used molecules, possible stimuli can be electrical energy (redox changes), electromagnetic energy (light) or chemical energy (change of pH value or addition of specific ions). Being an exceptionally novel field of science, starting in the mid 1980’s and just being rewarded with the Nobel prize of chemistry in 2016, the current applications of molecular machines are still rather few. However, a multitude of possible applications are being explored by the minute. Many mechanical devices can be mimicked, including rotors, oscillators, gears, paddle wheels, turnstiles, brakes, ratchets and gyroscopes.[1] One example of a more sophisticated system is a molecular motor[2] that can perform a 360° rotation by subsequent application of light and temperature, as can be seen in Figure 1. In another research group, four similar rotary systems have been combined on a larger molecule in order to create a four-wheeled nano-car that was able to move over a surface upon irradiation.[3]

In this project, azo dyes are used as molecular machines. Upon irradiation, the -N=N- azo group changes from a trans configuration to a cis configuration. As a consequence, the distance between the substituents R is shortened, resulting in a muscle-like contraction. The working principle of azo dyes as molecular machines can be seen in Figure 2.


References: [1] V. Balzani, A. Credi, M. Venturi: Molecular Devices and Machines – A Journey into the Nano World, Wiley-VCH, Weinheim, 2003. [2] N. Koumura, R. W. J. Zijlstra, R. A. van Delden, N. Harada, B. L. Feringa, Nature, 1999, 401, 152-155. [3] T. Kudernac, N. Ruangsupapichat, M. Parschau, B. Maciá, N. Katsonis, S. R. Harutyunyan, K.-H. Ernst, B. L. Feringa, Nature, 2011, 479, 208-211.

DEAs, or dielectric elastomer actuators, generally consist of thin elastic films coated with compliant electrode material on two opposing phases. That makes them flexible plate capacitors that deform due to Maxwell stress when applying a voltage across the electrodes. Since the elastic material is incompressible, applying said voltage causes the reduction of the film in thickness to result in an expansion in area. Stacking and bundling of multiple DEAs allows an actuation system to be adapted to different loading scenarios.Several materials are suitable for manufacturing DEAs. Electrodes can be made e.g. of metals, polymers, graphite or carbon nano tubes. Elastic dielectric materials in use for industrial applications are Polyacryl, Polyurethane or Polysiloxane. This project however focusses on the synthesis of a catcher-tag polymer as dielectric medium while using carbon nano tubes for the electrodes.

Manufacturing DEAs is possible in multiple ways. Thereby dipping, spin-coating and casting techniques are widely spread. For biomaterials, cartridges are more suitable for handling and dispensing the dielectric material. For the application of electrodes onto the elastomer, sputter and spraying techniques are widely used within the industry. Another option is the selective wetting process based on water-based solutions for when the polymer material is hydrophobic.

References: F. Nendel, S. Reitelshöfer: Conception of an infrastructure for the volumetric flow rate controlled supply of process gases and for the dynamic diversion of aerosol-flows for manufacturing flexible graphene electrodes. Institute for Factory Automation and Production Systems, (06/2017)