Engineering
Engineering cycle
Synthetic biology is a branch of engineering, as it applies the same principles to develop biological systems that perform specific functions. Much like the process an engineer uses to design a product, synthetic biology follows a series of iterative steps known as the Engineering Cycle. This cycle repeats until the system meets the desired specifications. In synthetic biology, the cycle continues until a biological system capable of achieving the desired function is identified.
The Engineering Cycle consists of four key phases: design, build, test, and learn. In the design phase, the target biological system and its intended function are specified. Literature research and modelling tools are especially useful here, as they can accelerate the design process by learning from previous results and simulating different constructs, saving time and resources in the lab. During the build phase, these designs are implemented in a target organism, or “chassis”, which is typically a strain of bacteria or yeast. Next is the test phase, where the system is evaluated to ensure it performs the desired function. Finally, the learn phase involves analysing the results: how much does the performance deviate from the expected outcomes, and what can be done next time to improve. Insights gained during this phase are then used to refine the initial design, and the cycle begins again.
Hemicellulose
The goal of our iGEM project of 2024, named KlothY, is to produce a unique, customizable and environmental-friendly kind of textile. This textile is going to be based on bacterial cellulose. But with the usage of bacterial cellulose, there will be a few challenges to face, due to its different properties in comparison to established textiles or cloths. Bacterial cellulose alone simply does not have the characteristics we imagine, when thinking about cloth in general. When creating mats of bacterial cellulose, it is easy to spot that they are very stiff and brittle [1]. Therefore, they cannot function as a comparable textile substitute. So, the logical consequence, to tackle these insufficient properties of a pure bacterial cellulose mat, is to increase their elasticity, flexibility and within also their endurance. In short, that is the main task of the hemicellulose subgroup and the problem our engineering cycle is trying to find a solution for. To achieve these property changes we want to increase the amount of hemicellulose, which is incorporated in our bacterial cellulose mats. The increased amount of hemicellulose within the mats will give the cloth more of the needed elasticity and flexibility [2, 3, 4]. So, in the end, the produced cellulose mats should, at least property-wise, get a lot closer to already established textiles or cloths.
Our Hemicellulose cycle:
Design
In order to achieve the goal of increasing the amount of expressed hemicellulose, we will boost the produced mass of xyloglucan, one kind of hemicellulose. Our whole plan consists of multiple steps. At first, we want to produce the xyloglucan. Therefore, we imagined a 5-gene strain, which is responsible for producing xyloglucan inside of the golgi-apparatus.
The named 5 gene-strain includes the following genes:
AtCSLC4 cellulose synthase like fam. C -Golgi membrane; xyloglucan glycosyltransferase; constructs glucose backbone
AtXXT3 xyloglucan 6-xylotransferase -Golgi membrane; xylose transferase; directs inside the membrane and associates with AtCSCL4
AtUGDH1 UDP-glucose 6-dehydrogenase -Cytosolic UDP-glucose to UDP-glucuronic acid
AtUXS3 UDP-glucuronic acid decarboxylase 3 -Cytosolic UDP-glucuronic acid to UDP-xylose
AtUXT1 UDP-xylose transporter 1 -Golgi membrane; UDP-xylose transporter
A model of the xyloglucan synthesis:
(Made with BioRender®)
The product will be built inside of the golgi. Its structure consists of a glucose backbone with the added xylose, forming xyloglucan. The assembled xyloglucan strings will intercalate with the bacterial cellulose mats, allowing them to change their properties.
(Made with BioRender®)
That part of our project would not have been coming to life without the work of Ronja Immelmann, Alexander Schultink and Balakumaran Chandrasekar. We received a plasmid from them containing the 5 genes needed for the xylose production inside of the golgi (CD108). Their work as well as their willingness to share a part of it with us, made it possible to work on solutions for our project.
The inducibility of the xyloglucan production represents the second step. To solve this problem, we want to test multiple promotors, because we need to be able to induce the xyloglucan production before growing bacterial cellulose mats.
In total there are three different variants:
1. Sc-pCYC1, a constitutive promoter for all 5 genes
2. pREV1-Z3EV, an orthologous inducible promoter
3. Sc-pGal1, a galactose inducible promoter for all 5 genes
About the orthologous inducible promoter (pREV1-Z3EV):
The inducible promoter pREV1-Z3EV allows us to induce our genes independently from our colouring-system via ß-estradiol. The Z3EV transcription factor is encoded by a gene coupled to a constitutive promotor.
The last step is going to be adding a leucine auxotrophy. Including this property to our plasmids will make us able to tell, which of our cells incorporated the plasmid. After finishing the specific plasmids of all needed constructs, the next goal is the expression of the complete xyloglucan pathway in Saccharomyces cerevisiae, an eucaryotic model organism. Reaching that goal, also consists of a few sub-steps.
Beginning with an assembly of a modular cloning set (MoClo). A MoClo is an efficient way to assemble many DNA parts into one functional plasmid. Since we planned 3 final plasmids with the 5 genes, promotors, homology regions, markers and a few more components, a MoClo portrays the best choice. These plasmids will be transformed and amplified in Escherichia coli.
Subsequently we want to transform S. cerevisiae and select specific colonies. That selection is going to be based on the leucine auxotrophy we included earlier. At last, and if everything else worked as desired, we want to measure and localize xyloglucan. By doing so, we will be able to make statements about the produced quantity of the xyloglucan and following also about the quality of our resulting mats.
Build
Our work for the subgroup started with the creation of all needed plasmids for our 5-gene-strain, the inducibility, as well as all of the fitting promoters, terminators and homology regions. The first plasmids were re-transformed from the iGEM distribution kits, containing multiple of our needed parts, and prepared for use at a later time. The general retransformation consists of the transformation, setting up liquid cultures, plating them and finally preparing the plasmids. A few of our needed parts, for example the 5 gene strain, were obtained from the institute of our PI Markus Pauly. Also, these plasmids were prepared and stored.
Test
Our work made it possible to test a few things. We were able to check and rate the quality, or more precisely the DNA concentrations, of our plasmid retransformations via the spectrometer. Our stock of L0 plasmids contained:
From the distribution kits.
The noted concentration of CD023 was measured before the fix.
The plasmid of the 5 gene strain (CD108) was prepared and shows the following concentrations:
While preparing these plasmids we got interrupted by one plasmid. CD023, which contained the OYC backbone. It was sequenced and did not fit the expectations. Multiple PCRs show the progress closer to our desired results.
The first gel electrophoresis:
(Its PCR-annealing happened at 60°C)
The second gel electrophoresis:
(Its PCR-annealing happened at 65°C and 70°C)
The third gel electrophoresis:
(Its PCR-annealing happened at 65°C and 72°C)
All of these gels refer to CD023a.
After fixing CD023, the samples were digested using SapI restriction enzymes and their concentrations were measured.
Through the digest of CD023 we obtained a few copies of CD024, which were sequenced and showed the expected sequences. To finalize CD024, we repeated its creation. Due to this repetition, we were sure, that CD024, as well as its basis CD023, would not contain any mistakes. The mentioned repetition also included a PCR, a gel electrophoresis and a DpnI digest.
The results of the gel electrophoresis:
It showed the desired results and its replication was stopped. To finish CD024, it was transformed and stored.
Learn
We stumbled over a few problems we were able to learn from. Primarily it is often useful to double check important parts of the construct. By doing so the occurrence of mistakes, especially in late phases of experiments or studies, can be minimized. Secondly, we learned, that it can be useful to test multiple PCR-annealing temperatures. Even little variance can lead to different results in quality
Sources: [1] Chanliaud E., Gidley M.J., (2002) In vitro synthesis and properties of pectin/Acetobacter xylinus cellulose composites https://doi.org/10.1046/j.1365-313X.1999.00571.x [2] Chanliaud E., et al., (2004) Mechanical effects of plant cell wall enzymes on cellulose/xyloglucan composites https://doi.org/10.1111/j.1365-313X.2004.02018.x [3] Whitney S.E.C., et al. (1999) Roles of Cellulose and Xyloglucan in Determining the Mechanical Properties of Primary Plant Cell Walls https://doi.org/10.1104/pp.121.2.657 [4] Chanliaud E., et al., (2002) Mechanical properties of primary plant cell wall analogues https://doi.org/10.1007/s00425-002-0783-8 Note: All pictures and tables above are made by team members and are saved in our elab-protocolls.
Inducible BC
To achieve our goal to have controlled activation of the Bacterial cellulose synthesis apparatus in Komagataeibacter xylinus. We went through multiple design cycles to achieve a genetically engineered strain where the bacterial cellulose production can only be activated by the presence of arabinose sugar.
Knockout Cycle 1
Design
We initially researched the enzymatic pathway for Bacterial cellulose in Komagataeibacter. In literature we found that the responsible genes BcsA, BcsB, BcsC and BcsD are the main enzymes involved in the synthesis of Bacterial cellulose. However, other Enzymes including BcsZ and BcsH also are necessary to ensure stable production. In addition we designed our Primers to have PaqcI restriction sites to be Add original plasmid maps for in- silico cloned knockouts
Build
We assembled our constructs by firstly by starting a genome DNA extraction in K. xylinus to have a DNA template to amplify the necessary homology regions for the constructs with PaqcI overhangs to later assemble.In addition we amplified the Level 0 backbone pSB1C30 with a Chloramphenicol resistance cassette and PaqcI to act as our backbone. And an ampicillin resistance cassette to select for later in final transformation in K. xylinus.
After the amplification of every fragment was complete. A Golden gate assembly with PaqcI and T4 ligase was done to assemble our constructs. After completion, our constructs were transformed in E. coli based on our DH5α transformation Protocol and plated on LB agar plates with both Cam and Amp inside for selection
Add planned Plasmid maps
Unfortunately some of the plates appeared to have dried out in the incubator they were placed in. Still out of 4 planned knockouts golden gates 3 of them were successful in growing colonies and initial colony PCR result showed that full plasmid was inside them
To verify the plasmid sequence. We did full plasmid sequencing through next generation Nanopore sequencing by Microsynth.
Add Sequencing results for knockouts
Sequence results showed that one of the knockouts actually was very similar to the planned plasmid we designed but in CD014 a homology region was missing. We are currently unsure why exactly. It was slightly unfortunate as both inducible constructs that were sequenced share the region affected but it may be due to human error during assembly. Due to time constraints a repetition of the transformation was not an option and we continued with CD015 plasmid construct
After verifying results through sequencing. We then transformed the previously prepared electrocompetent K. xylinus with the plasmid CD015. After 5 days visible colonies formed on plates
Add pictures of K. xylinus plate CD015
Colonies for cPCR results were picked and resulting gel indicate that knockout through homologous recombination was probably successfully integrated into the genome of K. xylinus
Picture : DNA electrophoresis gel pic. Layout of gel pic from left to right: ladder,2-7 replacing native constitutive promoter for the Bcs ABCD with paraBAD /inducible arabinose promoter and AraC and AraE genes through homologous recombination, 8-13 knockout of BcszH region through homologous recombination, 14-15 replacing native constitutive promoter for the Bcs ABCD with paraBAD /inducible arabinose promoter and AraC genes through homologous recombination.
Test
After positive control we really wanted to immediately characterise our transformed strains. However, due to time constraints we were not able to achieve an in depth characterisation. However, we still managed to do an initial comparative inoculation test by Preparing SOC media with 2% glucose and 1% Arabinose as well as SOC media with just 2% glucose added. We then added colonies from knockout strain CD015, inducible strain CD027 and WT for comparison.
Although the test is qualitative in nature it was mainly to assess if Bacterial cellulose is visible if induced and if so if there is a visual difference between performance of WT and knockout.
Learn
Uwe were able to achieve a viable transformation and through
1. Römling, U., & Galperin, M. Y. (2015). Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions. Trends in microbiology, 23(9), 545–557. https://doi.org/10.1016/j.tim.2015.05.005
Inducible Cycle 2
Design
We initially researched the enzymatic pathway for Bacterial cellulose in Komagataeibacter. In literature we found that the Enzymes BcsA, BcsB, BcsC and BcsD are the main enzymes involved in the synthesis of Bacterial cellulose. However, other Enzymes including BcsZ and BcsH also are necessary to ensure stable production. In addition we designed our Primers to have PaqcI restriction sites.
Build
We assembled our constructs by firstly by starting a genome DNA extraction in K. xylinus to have a DNA template to amplify the necessary homology regions for the constructs with PaqcI overhangs to later assemble.In addition we amplified the Level 0 backbone pSB1C30 with a Chloramphenicol resistance cassette and PaqcI to act as our backbone. And an ampicillin resistance cassette to select for later in final transformation in K. xylinus.
Picture 1: Table overview of necessary PCRs for both knockout and inducible BC
After the amplification of every fragment was complete. A Golden gate assembly with PaqcI and T4 ligase was done to assemble our constructs. After completion. Our constructs were transformed in e. coli based on our DH5 alpha transformation Protocol and plated on LB agar plates with both Cam and Amp inside for selection
Add planned Plasmid maps
Unfortunately some of the plates appeared to have dried out in the incubator they were placed in. Still out of 6 planned inducible constructs golden gates 3 of them were successful in growing colonies and initial colony PCR result showed that part of plasmid full plasmid was inside them
To verify the plasmid sequence. We did full plasmid sequencing through next generation Nanopore sequencing by Microsynth.
Add Sequencing results for knockouts
Sequence results showed that two of the inducible BC constructs showed almost identical sequences very similar to the planned plasmid we designed CD017 and CD027. Due to time constraints a repetition of the transformation was not an option and we continued with the named plasmid constructs
After verifying results through sequencing. We then transformed the previously prepared electrocompetent K. xylinus with the plasmid CD017 and CD027. After 5 days visible colonies formed on plates
Add pictures of K. xylinus plate CD015
Colonies for cPCR results were picked and resulting gel indicate that knockout through homologous recombination was probably successfully integrated into the genome of K. xylinus
Picture : DNA electrophoresis gel pic. Layout of gel pic from left to right: ladder,2-7 replacing native constitutive promoter for the Bcs ABCD with paraBAD /inducible arabinose promoter and AraC and AraE genes through homologous recombination, 8-13 knockout of BcszH region through homologous recombination, 14-15 replacing native constitutive promoter for the Bcs ABCD with paraBAD /inducible arabinose promoter and AraC genes through homologous recombination.
Test
After positive control we really wanted to immediately characterise our transformed strains. However, due to time constraints we were not able to achieve an in depth characterisation. However, we still managed to do an initial comparative inoculation test by Preparing SOC media with 2% glucose and 1% Arabinose as well as SOC media with just 2% glucose added. We then added colonies from knockout strain CD015, inducible strain CD027 and WT for comparison.
Learn
While we were ultimately succesful in generating a viable inducable Komagataeibacter strain. There were several points during the in silico cloning as well as the wetlabstage where improvement is indeed possible. The whole plasmid sequencing showed that in all E. coli with the inducible constructs the rbs was missing according to sequencing results
picture showing sequence alignment of CD017
picture showing sequence alignment of CD017
The cause could be that the rbs in combination with the araC and araE genes were stressing the E. coli as it increased the AraC amount in the cell. Resulting in the consistent removal of the ribosome binding site by the E. coli where the AraC gene was present as well. Luckiliy for us we had overhangs that were identical to the startcodon plus another base. Therefore there was a low chance it binds incorrectly overgoing the rbs. While we were lucky in that case future iGEM Teams should definitely not count on that and take this definitely into consideration if they want work with the construct
1. Römling, U., & Galperin, M. Y. (2015). Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions. Trends in microbiology, 23(9), 545–557. https://doi.org/10.1016/j.tim.2015.05.005 2. Mangayil, R., Rajala, S., Pammo, A., Sarlin, E., Luo, J., Santala, V., Karp, M., & Tuukkanen, S. (2017). Engineering and Characterization of Bacterial Nanocellulose Films as Low Cost and Flexible Sensor Material. ACS applied materials & interfaces, 9(22), 19048–19056. https://doi.org/10.1021/acsami.7b04927
Outlook
Outlook
With that we could eventually reduce the need for cellulase necessary to increase cell count and optimise carbon intake by targeting knockouts or overexpression for different genes and or clone an inducible promoter in front of the gene we want to up or down regulate, giving us even more control over the Bacterial cellulose synthesis. Through this we hope to push the scale in our favour to achieve a true sustainable product.
source
Dye Group
To achieve our goal to have coloured bacterial cellulose mats, we went through multiple design cycles in order to come closer to achieving a modular colouring system.
Dyeing of bacterial cellulose mats Cycle 1
Design
Due to chromoproteins being not heat-stable, we wanted to try dyeing our mats with different (substances). To achieve this, we settled on dried algae powder, beetroot, curcurmin and activated charcoal.
Build
We mixed the powdered pigments into the growth media (YPD) and inoculated it with K.xylinus from a plate. They were then set to incubation for 7 days at 30 degrees.
Test
All mats were autoclaved in dH2O and washed on an aggregator for 50 min in water and then dried on a drying rack. All mats except for the curcumin mat lost their colour.
Learn
Curcumin is suited as a natural pigment to use for staining bacterial cellulose. The bacterial cellulose mats are in this state not under the perfect condition of being dyed due to the intrinsic brownish colour.
Improvement of the dye assembly approach Cycle 2
Design
We initially designed all our in silico constructs for the expression of our chromoprotein constructs in E.coli with the terminator EF_TZ (BBa_J435371 )for our L1 constructs.
Build
We started our golden gate assembly with BBa_J435300 as the acceptor vector, our own designed constructs BBa_K5146000 and BBa_K5146020, a chromoprotein like aeBlue ( (BBa_K864401)) and the beforehand mentioned BBa_J435371.
Test
Running cPCRs with the white colonies that formed on xGal plates showed that either the E.coli had taken up multiple plasmids or only an uncut BBa_J435371 because it also contained an ampicillin resistance.
Learn
We learned that it extremely complicates the cloning and screening process and success if one of the L0 parts has the same resistance marker as the L1 you are aiming for. With that knowledge in mind, for all future reactions we switched to BBa_J428092 as a terminator because it has a chloramphenicol resistance and it does not pose the same problems as the previously mentioned terminator.
Dyeing of bacterial cellulose mats utilizing pigments Cycle 3
Design
Due to the first dyeing test with natural pigments being unsuccessful, we wanted to try a new approach, where the bacterial cellulose mat itself is better suited for dyeing to the colour. For the new dyeing approach, we wanted to try to utilise beta carotene as a pigment. From our experience gathered over the course of the project, we wanted to use a bacterial cellulose mat that is better suited for dyeing and has a lighter colour than incubated on YPD.
Build
We extracted a bacterial cellulose mat grown on YPG media which is way lighter in colour. The mat was washed in distilled water at 20 rpm for 50 minutes afterward, it was washed twice in 1% NaOH solution for 40 minutes each. The mat was then shaken in a beta-carotene solution for 2 hours and dried on parchment paper for 2 days.
Test
The bacterial cellulose mat did not desaturate throughout the drying.
Learn
Beta carotene is suited to stain bacterial cellulose mats furthermore due to the added washing steps the bacterial cellulose mat absorbs the colour better.
Property test
To cultivate our bacteria, K. xylinus, and thus our bacterial cellulose as a product, we first designed the composition of our culture media. For the media, we decided to set two variable parameters: the concentration of glycerol as our alternative sustainable carbon source and the concentration of xyloglucan as hemicellulose. The xyloglucan used in this project was partly obtained by extraction from tamarind seeds in our lab.
Mechanical Tests Cycle 1: Visual evaluation of the effects of hemicellulose and glycerol on the BC mat
Design
We planned to grow K. xylinus in “Hestrin-Schramm” (HS) media with varying hemicellulose and glycerol concentrations to test its effects on the properties of the bacterial cellulose produced.
In other experiments, we wanted to test how well Bacterial Cellulose mats would grow in “Yeast Peptone Sucrose” (YPS) and “Yeast Peptone Dextrose” (YPD) media.
Build
K. xylinus was grown in the media. After six days, the bacterial cellulose mats were harvested, washed and lastly dried.
Test
The mats were evaluated visually, where mats with higher Xyloglucan concentrations seemed to perform better relatively, as they retained their shape. Some of the mats in lower concentrations were also not extractable due to their flimsy attributes. Further, mats grown in YPD or YPS seemed to grow faster, thicker and more stable in general
Learn
All samples shrunk significantly after the drying process, and therefore an additional post-treatment or a change in the drying process needed to be devised. We learned that mats grown with xyloglucan were easier to extract and showed a more rigid structure, which supports that xyloglucan may enhance the physical properties of BC mats. The variation of glycerol concentration only resulted in a negligible effect on the pellicle formation.
As mats tended to grow better in YPD and YPS media, changing the media, we added our xyloglucan to would be the next step.
Mechanical Tests Cycle 2: Mechanical evaluation of the effects of Xyloglucan
Design
“Yeast Peptone Sucrose” (YPS) media with varying Xyloglucan concentrations were prepared. We also planned to test washing the BC mat with a 1% (w/v) NaOH solution hoping to remove media stains better than with ddH2O only and to maybe change properties. We also wanted to evaluate the possible property-enhancing effects of different drying methods like parchment drying and drying the mats in the drying cabinet.
Build
We grew mats with “Yeast Peptone Sucrose” (YPS) media of varying Xyloglucan concentrations. Different washing and drying methods were applied.
Test
Tensile strength tests were conducted on each sample to determine the effects of different xyloglucan concentrations, washing methods, as well as drying methods.
Learn
BC mat samples grown in YPS medium with 0.25% (w/v) xyloglucan concentration displayed the greatest tensile strength compared to other BC mat samples but showed reduced elasticity compared to mats without xyloglucan. Samples with xyloglucan concentrations showed reduced tensile strength as well as elasticity compared to 0% xyloglucan mats. This shows that other ways to increase the tensile strength and elasticity should be tested, as the addition of xyloglucan seems unviable. These could be things like reinforcing the textile by letting them grow into a net added to the growth media beforehand.
Previously, the washing steps were carried out with cold water but not NaOH. Treatment with NaOH enhanced both the elasticity and tensile strength of our BC mats.
In addition, drying in the drying cabinet increased the tensile strength and elasticity of the mats compared to parchment drying.
Chemical Tests Cycle 1: First HPAEC
Design
K. xylinus was grown in Hestrin Schramm (HS) media with varying hemicellulose concentrations (xyloglucan - 0.1~0.5%), to see if and how much of it was integrated into the mat. The amount of glycerol in the used HS media was also varied, to evaluate possible effects on the mats’ composition.
Build
K. xylinus was grown in the media. After six days, the bacterial cellulose mats were harvested, washed and lastly dried.
Test
The sugar composition of the produced BC mats was evaluated by HPAEC. Ribose was used as the internal standard.
Learn
During the HPAEC evaluation, we found that our samples already contained ribose. Another sugar needed to replace Ribose as the internal standard.
Chemical Tests Cycle 2: Second and Third HPAEC
Design
K. xylinus was again grown in HS media with hemicellulose but this time only with 0.5% and 0% xyloglucan which served as a contrast, as we found in the first engineering cycle of the mechanical tests, that mats grown with higher concentrations of Xyloglucan seem to be more rigid.
Build
After six days, the bacterial cellulose mats were harvested, washed and lastly dried. In addition, BC mats grown in 0% xyloglucan but later washed in a 0.5% xyloglucan solution were prepared.
Test
The sugar composition of the produced BC mats was evaluated by HPAEC. Ribose was used as the internal standard.
Learn
Xylose was absent in the BC mat grown without Xyloglucan (0% XG). A considerable amount of xylose was found in the mat washed with 0.5% (w/v) XG solution (0% XG washed in 0.5% XG) and a relatively high amount in the mat grown with 0.5% Xyloglucan (0.5% XG), which means, that Xyloglucan was incorporated in the Bacterial Cellulose mat.
Hardware Cycle 1: Designing the first machine
Design
We designed a property testing machine to be able to run our own mechanical tests on our produced textiles.
Build
We built the designed machine with wood as our main building material.
Test
We tested if tear tests would be possible with the construction by ripping apart pieces of cardboard, held by self-built clamps, with the upper one being connected to a string which was pulled upwards by hand.
Learn
We learned that our designed test could work if improved further, but when we showed the machine to one of our PIs, Prof. Markus Pauly, he made us aware of the problem, that we wouldn’t be able to properly clean the machine if something happened in the lab, meaning we had to go for a non-porous material, to build our machine with.
Hardware Cycle 2: Designing the improved machine and first winding mechanism
Design
We redesigned the initial property testing machine’s design, with galvanised sheet metal as our main building material, as it could be cleaned without problems. We also designed a winding mechanism to substitute for pulling the string/cable by hand.
Build
We built the frame for the machine as well as the first winding mechanism made of LEGO to substitute for pulling by hand (see picture). For the clamps, we reused the ones from the previous machine.
Test
We tested the winding mechanism and realised that it wasn’t able to properly wind up the string/cable. The stability of the frame was evaluated in the same way as the wooden machine before.
Learn
We learned that we would have to redo the winding mechanism with more area to wind the string on. The frame worked as intended.
Hardware Cycle 3: Improving the winding mechanism
Design
Based on a construction by the iGEM team from GreatBay_SZ 2019, we redesigned the winding mechanism.
Build
We built the new winding mechanism (see picture) and also connected it to the frame for the tests this time.
Test
We tested the winding mechanism by ripping apart cardboard with it, which worked well, but faced problems when we tried to go for textiles, which we couldn’t rip apart. To quantify the force output, we held against the pull of the motor with a Newtonmeter and found that the force output was only about 6 N.
Learn
We learned that our winding mechanism worked, but that our motor was too weak, meaning we had to take a stronger one.
Hardware Cycle 4: Increasing the force output
Design
We redesigned our winding mechanism to fit two stronger motors, which could then move simultaneously, increasing the force output.
Build
We built the new winding mechanism with two bigger LEGO motors (see picture). As this couldn’t be used with the regular control panel, we also wrote code to be able to make it usable (See software)
Test
We tested the winding mechanism and code by ripping apart the textile which we had failed to rip apart with the previous construction. As this worked, we measured the force output which was above what we could measure with our Newtonmeter which could only measure up to 30 N.
Learn
We learned that our winding mechanism worked, but that our measuring range was too small, which would have to be increased with stronger Newtonmeters for example. We also learned from one of the tests, where the upper clamp flew away when the textile ripped, that this was a safety hazard which had to be dealt with.
Hardware Cycle 5: Integrating Safety Mechanism
Design
We designed a safety mechanism so that our upper clamp wouldn’t fly off when a textile rips apart.
Build
We integrated the safety mechanism by adding a WAGO clamp to the Newtonmeter’s hook.
Test
We tested the safety mechanism which worked at lower forces but less above the detection limit.
Learn
We learned that our safety mechanism would have to be improved further to allow for higher force outputs.