When we constructed our biobricks we used sequences from iGEM´s registry, our supervisors and the online program Benchling database. When all the sequences had been gathered, we put them together with Benchling. After the final touch was made, the biobricks were sent to IDT who transformed our constructed sequences into DNA.
Our plasmids were designed to include different options for overexpression of chaperones. This was done by connecting each chaperone to an individual promoter and promoter region which enables overexpression both in individual chaperones, but also overexpression of different combinations of chaperones. Our method of assembly was NEB Hi-Fi DNA assembly. To enable this we created synthetic overlaps between the chaperones and the vector pSB_1C3. Each chaperone was also equipped with an individual terminating sequence to prevent unwanted downstream protein expression. We also designed a second plasmid containing the different fusion proteins. pSB_1A3 was chosen as the vector to enable a second selection with both ampicillin and chloramphenicol to ensure correct transmission of plasmids. The fusion protein consisted of two parts, a aggregation prone part (either amyloid beta 42 or TauN0R4) and a fluorescent part (mNeongreen or eGFP). Amyloid-beta 42 was chosen since aggregations of it is thought to be the cause of Alzheimer's disease (1). Although the isoform TauN0R4 is not fully confirmed to be a specific cause of Alzheimer's disease. Tau aggregation is still seen in the disease and the R segment is thought to be the part that aggregates. Therefore TauN0R4 was chosen over its counterpart TauN0R3 (2). When choosing the fluorescent part two factors were taken to account. On one hand we wanted to work with a well characterised protein but on the other hand we wanted a fluorescent protein that did not prevent the expression of our aggregation prone proteins, therefore the two fluorescent proteins we chose was eGFP and mNeongreen. eGFP is a well used and characterized protein and mNeongreen is about 1,5 - 3 times as bright as eGFP and has proven to be a good fluorescent tag in fusion proteins (3). All the aggregation prone fusion proteins carried a histidine tag to enable IMAC analysis (4). A TEV site was placed after the histidine tag so to enable a IMAC purification (4). We also designed a GS-linker between the fusion proteins to make them more motile (5). One big difference between the fusion proteins containing TauN0R4 and Amyloid-beta-42 is that Amyloid-beta-42 is placed C-terminally and TauN0R4 is placed N-terminally this is do to the fact that Tau starts the aggregation as soon as it gets translated and we wanted our protein to be as hard to express as possible (2).
Press here for a closer look at our parts.
To verify a successfully assembled biobrick we used several different methods both for DNA analysis and protein analysis.
Gel electrophoresis with a 1.3% agarose gel was used to control the size of the assembled fusion proteins and chaperone systems. This gave a hint on whether the assembly products were correctly assembled and if all parts in the assembly reaction had ligated or not. Because we already knew the size of our designed constructs we could easily see when the assembled products had the right number of nucleotides. If not all the parts in the assembly had ligated this would be shown as lighter bands on the gel.
To verify our assemblies, one of our PIs helped us send our DNA to GATC Biotech in Germany to get a full sequencing.
There are several different kinds of molecules called fluorophores that are prone to fluoresce, which is the outcome of a three-step process. The first step involves that the fluorophore absorb energy in the form of photons from a lightsource. Under the second step the fluorophore changes its’ conformation and some energy goes to molecules in its’ surroundings. During this time the fluorophores will lose some of the excitation energy. In the last step a photon is released from the fluorophore and goes back to its’ ground state. Due to the energy lost in the second step the photon will have a lower energy and a bigger wavelength. Therefore a different color than the absorption light will be detected (6). In our project we decided to use fluorescence as a marker of detection of our fusion proteins and were thus able to detect the protein concentration. In future science this could be used to analyse Amyloid Beta proteins where some have the fluorescence protein and some does not. In this way scientists will get a new view of how Amyloid Beta protein aggregates.
We analyzed the protein Amyloid-Beta fused with mNeonGreen with a plate reader. mNeonGreen is excited at 506 nm and emits light at 517 nm (3). The plate reader enables quantification of the fusion protein. We did not lyse the cells before the plate reading, we simultaneously measured the optical density (O.D) of each well. The O.D measurement made the amount of protein relative to the amount of bacteria in each well. To further analyze our protein we have used western blot. Western blot is a method where the proteins are denatured with SDS (Sodium dodecyl sulfate ) and run through a polyacrylamide gel electrophoresis. The gel is then washed and antibodies are applied to the gel. We used a primary antibody 6E10 from mouse that is specific to amyloid beta protein. We used a secondary antibody from rabbit that binds to the primary mouse antibody. The secondary antibody is conjugated with alkaline phosphatase and addition of CDP-star chemiluminescent substrate allows detection by chemiluminescent signals.
Positive and negative controls were used in the methods to ensure reliable results and to simplify troubleshooting in case of unsuccessful results. This was especially important when we ran a gel, made a culture of bacteria that had gone through a heat shock or electroporation and when taking values on the micro plate reader.
We wanted to find out which temperature would give rise to the most optimal expression of our proteins Amyloid-Beta and Tau with the different chaperone combinations. The plan was to try four different temperatures, 37°C, 30°C, 15°C and 8°C.
Finding the right amount of inducing agents for each promoter was also important for finding the optimal protein synthesis. By testing bacteria with different concentrations of the inducing agents, including non-induced bacteria, an approximative optimal concentration could be found. Promoters can sometimes leak and to detect this we took values on non-induced bacteria as well. We could also get a value of fluorescence between induced and non-induced bacteria. To find the optimal concentration to induce our promoters with the respective inducing agents for each promoter, we tested different concentrations of these. Some samples were controls and did not get induced. This to see if the promoters are leaky and to see the difference in fluorescence between induced and non-induced proteins.
The third parameter of induction we wanted to study was time. We wanted to see which time would give rise to the most optimal expression for our proteins and thereby the most fluorescens. The plan was to increase the amount of hours in relation to lower temperatures. The samples which would incubate at 37°C would be induced for 6 h, the samples incubating at 30°C would be induced for 12 h, the samples incubating at 15°C would be induced for 24 h and those samples incubating at 8°C would be induced for 48 h.
Our plan for the chaperone plasmid was, as already mentioned, to find the best combination of the chaperones in the best possible environment for maximum fluorescent. If this had succeeded, our plan was to amplify just that part of the superduper plasmid in a PCR and then create a new biobrick of that sequence. The purpose of our model was to enable faster information about what temperature, amount of inducing agent and time that would be the best optimization for protein expression.
1. Findeis MA. The role of amyloid β peptide 42 in Alzheimer’s disease. Vol. 116, Pharmacology and Therapeutics. 2007. p. 266–86. 2. Fitzpatrick AWP, Falcon B, He S, Murzin AG, Murshudov G, Garringer HJ, et al. Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature [Internet]. 2017;547(7662):185–90. Available from: http://www.nature.com/doifinder/10.1038/nature23002 3. Shaner NC, Lambert GG, Chammas A, Ni Y, Cranfill PJ, Baird MA, et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods [Internet]. 2013;10(5):407–9. Available from: http://www.nature.com/doifinder/10.1038/nmeth.2413 4. Bornhorst BJA, Falke JJ. Reprint of: Purification of Proteins Using Polyhistidine Affinity Tags. Protein Expression and Purification. 5. Chen X, Zaro JL, Shen WC. Fusion protein linkers: Property, design and functionality. Vol. 65, Advanced Drug Delivery Reviews. 2013. p. 1357–69. 6. ThermoFisher Scientific. Fluorescnece Fundamentals [Internet]. ThermoFischer Scientific [Cited 1 Nov 2017] Available from: https://www.thermofisher.com/se/en/home/references/molecular-probes-the-handbook/introduction-to-fluorescence-techniques.html 7. Jeong H, Kim HJ, Lee SJ. Complete Genome Sequence of Escherichia coli Strain BL21. Genome Announc [Internet]. 2015;3(2):e00134-15. Available from: http://genomea.asm.org/lookup/doi/10.1128/genomeA.00134-15
