Team:Aachen/Engineering
Engineering Success
A small step for us, a giant leap for M.A.R.S.
In our project, we realized that some of the experiments we planned did not work within the first try. Therefore, we had to evaluate the problems we faced and had to come up with new solutions. Even if something works, there is always the opportunity to improve further experiments.
Search for the anchor peptides
One of our project's central laboratory goal was to find an anchor peptide that binds to the magnetic particle as well as a second anchor peptide that binds to our liposomes.
So, we first had to synthesize liposomes. After some research, we decided to use a mixture of PC, DOTAP, and DOPC to have slightly positively charged liposomes for better bacteriorhodopsin integration[1]. The desired mixture of 30% DOTAP, 35% DOPC, and 35% POPC did not work; there were no liposomes visible. We then tried liposomes consisting of only POPC first. POPC worked with different buffers Tris-HCl 30 mM, pH 7.4(protocol) and Sorbitol 1 M(protocol) . DOTAP and DOPC also worked for themselves with Tris-HCl 30 mM, pH 7.4(protocol) . After that, we tried a mixture of 80% POPC and 20% DOTAP to get closer to the desired positively charged composition, which also worked.
With the formed liposomes, we could start the binding tests with different anchor peptides. First binding tests were performed with POPC liposomes because the formation of these was the most reliable. Our first approach was to try centrifugation through a molecular filter in order to separate liposomes from unbound anchors. But the centrifugation seemed to destroy the liposomes, which made us think about alternatives.
Another idea was to try size exclusion chromatography. This method has the risk that the liposomes interact with the column material and break. We therefore decided to try out a gentler approach. With dialysis, we could keep the bigger liposomes inside the dialysis tube and lower the concentration of unbound anchors because they are small enough to get through the dialysis tube. Due to the anchor peptides' size at around 35 kDa (anchor peptide + eGFP), we tried a dialysis tube with a molecular cut off 50 kDa. After a few tries, we had to realize that the anchors could not get through the tube membrane. Evaluating the problem, we concluded that they might create a hydrate shell, making them way bigger. New tests should be performed with a molecular cut off 100 kDa or even 300 kDa.
Due to the limited time left in the lab, we decided to first look for the best binding candidate with just qualitative tests. With the most promising candidates further tests with dialysis tubes should be performed. The new tests were performed with low anchor concentrations to see if the liposomes or the whole background would show fluorescence first. So far only LCI showed a fluorescence of the liposomes. Therefore, more tests with LCI should be performed.
Another idea was to try size exclusion chromatography. This method has the risk that the liposomes interact with the column material and break. We therefore decided to try out a gentler approach. With dialysis, we could keep the bigger liposomes inside the dialysis tube and lower the concentration of unbound anchors because they are small enough to get through the dialysis tube. Due to the anchor peptides' size at around 35 kDa (anchor peptide + eGFP), we tried a dialysis tube with a molecular cut off 50 kDa. After a few tries, we had to realize that the anchors could not get through the tube membrane. Evaluating the problem, we concluded that they might create a hydrate shell, making them way bigger. New tests should be performed with a molecular cut off 100 kDa or even 300 kDa.
Due to the limited time left in the lab, we decided to first look for the best binding candidate with just qualitative tests. With the most promising candidates further tests with dialysis tubes should be performed. The new tests were performed with low anchor concentrations to see if the liposomes or the whole background would show fluorescence first. So far only LCI showed a fluorescence of the liposomes. Therefore, more tests with LCI should be performed.
First results
The first test with the magnetic particles turned out well. We identified MBP-1 to have the best binding due to the highest fluorescence. Under the same conditions a higher fluorescence means that more anchor peptides are bound to the target surface.
eGFP (negative control)
LCI
MBP-1
Cg-Def
TA2
before washing
after washing
With these first results we concluded that a possible biadhesive peptide would be a fusion protein of MBP-1 and LCI. Both anchor peptides are connected through a stiff spacer Domain Z. This construct is able to connect polystyrene coated magnetic particles to POPC liposomes. Further test should be performed with different lipid compositions, especially with the desired slightly positively charged lipids. The whole fusion protein can then be expressed and binding of both complexes can be checked. This could be done through the use of common bar magnets to see if the liposomes would also be separated.
As an outlook for further experiments, it would be recommendable to find an anchor that binds to the magnetic particle but not to the liposomes and an anchor peptide that binds the liposomes but not the magnetic particle. This can ensure that only liposomes and magnetic particles are connected and not two magnetic particles or two liposomes with each other. Therefore, experiments should be performed with the chosen anchor peptides and both of the target surfaces. This experiment can be considered successful if, and only if, each anchor peptide binds only to its expected target surface.
As an outlook for further experiments, it would be recommendable to find an anchor that binds to the magnetic particle but not to the liposomes and an anchor peptide that binds the liposomes but not the magnetic particle. This can ensure that only liposomes and magnetic particles are connected and not two magnetic particles or two liposomes with each other. Therefore, experiments should be performed with the chosen anchor peptides and both of the target surfaces. This experiment can be considered successful if, and only if, each anchor peptide binds only to its expected target surface.
References
[1] Ritzmann N, Thoma J, Hirschi S, Kalbermatter D, Fotiadis D, Müller DJ. Fusion Domains Guide the Oriented Insertion of Light-Driven Proton Pumps into Liposomes. Biophys J. 2017 Sep 19;113(6):1181-1186. doi: 10.1016/j.bpj.2017.06.022. Epub 2017 Jul 8. PMID: 28697898; PMCID: PMC5607040