Team:Aachen/Experiments
Experiments in the Lab
Here's how the magic happened! In the Institute of Biotechnology and the FZ Jülich
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 and FZ Jülich, 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 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.
Due to the possibility of working at two different locations, namely the Institute of Biotechnology and FZ Jülich, 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 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.
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]. 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.
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]. 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]. 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.
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
and
Media
Proteins
and
Integration
and
Integration
Liposomes
and
Binding
and
Binding
Proof
of
Concept
of
Concept
Results
Here you will find the entirety of our lab results (failures included). These consist of:
For further information about the experiments please refer to our Lab Notebook
- Liposome formation
- Liposome and magnetic particle binding tests
- Protein purification and integration
- Application in a relevant context
For further information about the experiments please refer to our Lab 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]
The compositions that worked are:
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
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.
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.
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.
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
after washing
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
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.
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] 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.
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] 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.
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