To evaluate the binding affinity of our interactors to progerin, we use Microscale Thermophoresis (MST) on the Monolith X system. MST allows sensitive detection of molecular interactions in solution and provides dissociation constants (Kd) that reflect affinities under near-physiological conditions. From the resulting dose–response curves, Kd values are extracted, offering quantitative insight into the strength and specificity of each interaction.
Cell-free transcription-translation (TXTL) is a powerful system that enables protein and peptide expression directly from DNA, without the need for living cells. It relies on bacterial extracts containing ribosomes, tRNAs, polymerases, nucleotides, amino acids, and an energy-regeneration mix. Once a DNA template is added, the extract transcribes it into mRNA and immediately translates it into the corresponding protein.
In our project, TXTL is used to produce the synthetic peptides designed as potential interactors of progerin. Instead of cloning each sequence into cells and waiting for bacterial cultures, we can express them directly in vitro. This approach saves time and allows rapid testing of multiple peptide candidates.
Concretely, we ordered plasmids carrying the peptide DNA sequences from IDT. Shipped in lyophilized form, they are resuspended in nuclease-free water and directly added to the TXTL reaction. After a few hours of incubation, the system produces the desired peptides. These peptides are then purified and tested with the Monolith X instrument, which quantifies their binding affinity to progerin. This workflow not only accelerates the design–build–test–learn cycle of our project but also provides preliminary experimental validation of our interactors before moving to mammalian cell models.
Reagents (Syn-Xpress™ SILVER mix and DNA) were thawed on ice and handled carefully, avoiding vortexing. A 50 µL reaction was prepared with 40 µL of Syn-Xpress™ SILVER mix (final 1×) and a volume of synmEGFP plasmid DNA sufficient to achieve a final reaction concentration of 15 ng/µL, or nuclease-free water for the negative control. The volume was adjusted to 50 µL with nuclease-free water and mixed gently to prevent bubble formation.
Reactions were sealed in tubes with sufficient headspace for oxygenation and incubated at 150 rpm for 6 h with shaking. Positive and negative controls were incubated at 20 °C, while an additional positive control was incubated at 30 °C. At the end of incubation, samples were placed on ice.
mEGFP expression was measured with a Thermo Scientific™ Varioskan™ LUX Multimode Microplate Reader. Fluorescence values (RFU, Relative Fluorescence Units) were:
- Positive control at 30 °C: 260 RFU
- Positive control at 20 °C: 859 RFU
Since the recombinant progerin ordered from Diatheva has not yet arrived, we began by expressing and testing SpyTag and SpyCatcher. These well-characterized proteins form a stable covalent interaction, making them an ideal model system. Working with them allowed us to familiarize ourselves with the Monolith X instrument and validate the workflow before moving to experiments with our designed interactors and progerin.
Based on the yields obtained from control reactions, we expressed the peptides at 20 °C. Each CFPS reaction contained 40 µL of Syn-Xpress™ SILVER mix (final 1×), 15 ng/µL of plasmid DNA, and nuclease-free water to a final volume of 50 µL, mixed gently to prevent bubble formation.
The resin (HisPur™ Ni-NTA, 50 µL effective volume) was washed with Milli-Q water and then with PBS (centrifugation 5 min, 3000 rpm each). The 50 µL TXTL reaction was loaded onto the resin and incubated for 30 min at room temperature with shaking to promote binding.
The resin with bound proteins was transferred into a spin column and processed sequentially:
- Flow-through (FT): collected after washing with PBS 1× (300 µL);
- Wash (W1): obtained after an additional wash with PBS 1× (200 µL);
- Elution: proteins were eluted twice with PBS 1×, pH 7.7 containing 500 mM imidazole (200 µL each), yielding fractions E1 and E2;
To assess the presence of the recombinant proteins SpyTag and SpyCatcher, we performed SDS-PAGE on samples collected during purification. The results, however, were unsatisfactory: the expected His-tagged bands were faint or obscured, and the gel displayed multiple background bands. These likely originated from endogenous proteins in the cell-free synthesis system that were retained during early purification steps.
This nonspecific binding was probably due to an excessively high resin-to-protein ratio, which promoted interaction of non-target proteins with the affinity matrix. As a result, the purity of the eluted fractions was compromised, making it difficult to identify the target proteins with confidence.
To address these issues, we plan to optimize the purification protocol by adjusting the resin volume relative to the protein concentration in future experiments. This should help reduce nonspecific binding and improve the clarity of SDS-PAGE results, allowing us to better evaluate the expression and purity of our target proteins.
To improve retention and resolution of these proteins, we are repeating the experiment with a more suitable gel matrix. Two controls will also be included: one with the reaction mixture lacking expressed protein and another with resin alone. Detection will be refined by anti-His Western blotting, as both SpyTag and SpyCatcher are His-tagged.
Here is the image of the gel:
| Interactor | Code |
|---|---|
| Interactor 1: 62aa11_1 | BBa_254PC42U |
| Interactor 2: LOGO | BBa_252ADNO1 |
| Interactor 3: n80_02 | BBa_25KT4K90 |
| Interactor 4: Rank_15 | BBa_25E35V1M |
| Interactor 5: Rank_21 | BBa_253S6Z46 |
| Linker-SpyTag | BBa_25BEEQ7F |
| SpyCatcher | BBa_259R437F |
We used Saccharomyces cerevisiae as a cellular model to study the toxicity of progerin underlying the Hutchinson–Gilford Progeria Syndrome. To assess the impact of this protein on yeast cells, several experiments were performed.
In the experimental process we used pYES2 expressing progerin sequence optimization (BBa_25NDL8N0) for the expression in yeast cells by selecting proper codon usage. This plasmid expresses progerin under the galactose-inducible promoter GAL1 which allows controlled expression of the progerin sequence. It carries a selectable marker (uracil synthesis pathway) which enables growth selection in transformed S. cerevisiae strains with auxotrophy for uracil.
Bacterial transformation and plasmid extraction
The progerin-expressing plasmid was initially transformed into competent bacterial cells in order to amplify its quantity. We followed the standard manufacturer’s protocol and worked with 5 μL of plasmid and 25 μL of competent cells E. coli TOP10.
Transformed cells were incubated overnight at 37oC on solid LB medium supplemented with ampicillin (final concentration = 0.1 mg/ml). The following day colonies were isolated for plasmid extraction and further analysis.
Plasmid DNA was extracted with a miniprep Kit based on alkaline lysis and silica column purification, and quantified using a NanoDrop spectrophotometer. Additionally, the extracted plasmid DNA was checked by gel electrophoresis.
Chemical transformation
Once confirmed that the plasmid was correctly expressed in bacterial cells, confirmed by both agarose gel electrophoresis and Sanger sequencing, we proceeded with the Chemical transformation of yeast cells in order to obtain our transformed yeast culture. The efficiency of this method relies on ensuring a sufficient number of competent cells, thus increasing the probability of successful plasmid uptake. We used the CEN.PK strain, which was inoculated and grown overnight in rich YPG medium at 28°C with agitation. Each transformation required 2 mL of overnight culture. Pelleted cells were resuspended with all the necessary reagents, along with 1000 ng of plasmid DNA. After transformation, cells were plated on minimal solid SD Glu - URA medium. Following 2 days of incubation at 28oC we were able to select only the cells that had successfully taken up and expressed the plasmid.
Protein extraction, quantification, SDS-PAGE and Western blot
To verify protein expression in the transformed yeast cells, we incubated them in liquid SD Gal -URA medium overnight at 28oC, allowing galactose to induce progerin expression and provide sufficient protein for analysis. The following day, we measured the OD of each inoculum to calculate the volume required to achieve a final OD of 5. For protein extraction we applied the Trichloroacetic acid (TCA) method combined with glass beads for cell lysis. The extracted proteins were solubilized in Laemmli Sample Buffer 1X (LSB), the pH was adjusted with Tris-HCl (pH 8.8), and the samples were denatured at 95°C for 5 minutes. Proteins in the supernatant could then be stored at -20oC or used immediately.
Protein quantification is a crucial step in protein analysis. The Bradford assay is a colorimetric method for protein quantification based on the binding of Coomassie Brilliant Blue G-250 dye to proteins. We applied this method to quantify our samples, ensuring that the equal amounts of protein were loaded from each sample in order to obtain comparable results within each well.
After extraction and quantification, we proceeded with SDS-PAGE (Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis). In this method, Sodium Dodecyl Sulfate (SDS) denatures proteins and imparts a uniform negative charge, enabling their separation according to molecular weight during electrophoresis. At the end of this process, the gel was ready for protein transfer.
Protein transfer was performed onto a PVDF membrane (previously activated in ethanol) using a semi-dry transfer system: 20 minutes at 16 mA (through the stacking gel) and 1h at 24 mA (through the running gel). Successful protein transfer was confirmed by Ponceau staining, which revealed visible bands.
The membrane was then blocked with 5% milk in TBS-T for 1h at room temperature under gentle stirring. After blocking, the membrane was washed with TBS-T to remove residual milk and subsequently incubated with the primary antibody.
Our part BBa_25NDL8N0 contains a myc epitope tag, designed for Western blot detection. Therefore, the membrane was incubated with a mouse anti-myc primary antibody diluted 1:1000 in 1% BSA in TBS-T for 1h and 30 min at room temperature, followed by three washes with TBS-T (5 minutes each, under gentle stirring).
A freshly prepared secondary antibody, diluted 1:3000 in 1% BSA in TBS-T, was then applied for 1h at room temperature under stirring. After incubation, the membrane was washed three times in TBS-T (5 minutes each) and once in TBS for 5 minutes.
Detection was performed using luminol solution to activate the peroxydase activity of the secondary antibody, and the signal was visualized using a Uvitec imaging system.
The results confirmed protein expression, indicating that our yeast culture successfully expressed progerin.
Vitality tests
Once we obtained significant results for the expression of progerin in CEN.PK we tested some independent colonies to gain a better understanding of how the mutant protein can affect the phenotype of S. cerevisiae.
We aimed to monitor the growth of yeast cultures in liquid and solid glucose and galactose medium to study the effect of progerin on the cell growth.
Since the three pYES_empty colonies and the five pYES_progerin colonies used for the Western Blot had been restreaked and stored at 4°C for preservation, we inoculated them into 10 mL of SD-GLU –URA medium.
When the yeast cells had reached the stationary phase in a selective minimal medium we started to set our growth curve analyses.
To standardize the initial cell density and ensure comparable growth curves across various conditions, the optical density (OD) of each overnight culture was measured at 600 nm. The volume needed to achieve an initial OD600 of 0.15 in 15 mL of fresh medium was calculated using the following formula: ODculture × Vinitial = ODfinal × Vfinal
Each 15 mL culture was transferred into a 50 mL flask to ensure proper aeration and robust growth.
We worked in duplicate with two strains in two different media (SD Glucose 2% -URA and SD Galactose 2% -URA) both for the control and the samples to test. After inoculation, cultures were incubated at 28 °C with shaking and monitored over time.
A volume of 800 µL was taken without dilution to measure the initial OD of the cell culture. This was used to verify that all cultures started at a standardized OD600 of 0.15. For the first measurements in the afternoon, after approximately 6 hours of incubation, samples were diluted in a 1:2 ratio for both galactose and glucose cultures. The same dilution (1:2) was applied for the following measurements (after another 6 hours) for galactose cultures, while glucose cultures were diluted 1:10. For the subsequent measurement periods (the following afternoon and morning), all cultures were diluted 1:10 before OD readings.
Although the overall growth trends were not sharply distinct, a clear difference became evident after 24 hours between the progerin-expressing cultures and their respective controls.
Simultaneously we worked on the spot test. The spot test allows us to compare the growth of the same cultures under different conditions. Up to eight cultures can be tested at the same time, with each culture plated in four serial dilutions (1, 10-1, 10-2 , and 10-3) to better assess differences in growth. Each dilution is prepared from a culture standardized to an OD of 1, meaning that the first dilution corresponds to an OD 1 cell concentration.
Plating was performed by depositing a drop of each culture onto the pre-designed array on the plate. Plates are then incubated at 28°C for three days, and photographs are taken daily to capture the full progression of growth.
All of the five colonies containing pYES_progerin were tested and we used pYES_empty as control. We measured the optical density (OD) of each culture to calculate the appropriate dilutions, standardizing the starting concentration to OD = 1 for the first spot.
We then calculated the ml needed to achieve this OD in 1ml of water. Then a series of four 1:10 serials dilutions were prepared and we pipetted 4 µl of volume from each dilution to compare the growth of the same cultures on different mediums.
In order to evaluate the impact of the protein expression on cells, growth differences across dilutions and different media conditions are analyzed. Every 24 hours, pictures of the plates were taken to visually track growth and confirm the trends observed in OD measurements. At 24 and 48 hours, all strains grew similarly on glucose medium, showing colony formation regardless of the presence of the plasmid. This indicates that under repressive conditions, the presence of the plasmid does not affect the viability of the yeast cells.
In contrast, on galactose plates, a clear difference between two strains could be seen over time. Colonies carrying the empty pYES2 maintained consistent growth, indicating that galactose itself does not inhibit growth. In contrast, yeast carrying the pYES_Progerin plasmid showed progressively reduced growth on galactose plates compared to those growing in glucose and to the empty vector controls. This growth reduction was more noticeable at 72 hours, where the density and size of the colonies expressing progerin decreased significantly. These results confirm that the expression of progerin under galactose-inducing conditions exerts a toxic effect on yeast cells.
S. cerevisiae provides a convenient and cost-effective system to study protein-protein interactions using the Yeast Two-Hybrid (Y2H) assay. This approach allows us to test the interaction between two proteins via the use of two plasmids: one encoding the “prey” protein and the other encoding for the “bait” protein. In our experiments, we used the commercially available plasmids pGADT7 and pGBKT7. The pGAD plasmid expresses the activation domain (AD) and serves as the prey vector, whereas pGBK expresses the DNA-binding domain (BD) and serves as the bait vector.
The genes encoding the proteins of interest can be cloned into these plasmids in frame with the AD or BD domains, enabling the system to assess potential protein-protein interactions.
When both plasmids are co-transformed into the same S. cerevisiae cell, an interaction between the two proteins brings the BD and AD domains into proximity, reconstituting a functional transcription factor that activates a reporter gene, allowing yeast growth on selective medium.
The experimental procedure for performing Y2H consists of two main steps:
- Co-transformation of the bait and prey plasmids into the same yeast cell;
- Spot test assay on selective medium to assess activation of the Y2H reporter pathway
Plasmids for Y2H
The plasmids employed in Y2H are:
- pGBKT7: expresses the DNA-binding domain (BD) fused to a myc epitope and serves as the bait plasmid. Its nutritional selection relies on tryptophan auxotrophy. In bacteria, this plasmid can be selected by its Kanamycin resistance;
- pGADT7: expresses the activation domain (AD), fused to a HA epitope and a Nuclear Localization Signal (NLS), serving as the prey plasmid. Its nutritional selection relies on leucine auxotrophy. In bacteria, this plasmid can be selected by its ampicillin resistance
In our experiment, the report pathway used is the HIS3 pathway. Accordingly, the selective medium is SD Glucose -Leu -Trp -His +3-AT. The addition of 3-AT (3-aminotriazole), an inhibitor of histidine synthesis, is necessary because activation of HIS3 is leaky, and it strengthens selection for true positive interactions.
Cloning progerin for Y2H
At the start of our Y2H experiments, we first cloned the full-length sequence of progerin (in this case, a non-RFC10-standardized sequence) into both the Y2H plasmids, pGADT7 and pGBKT7.
Our aim was to assess whether Y2H could be applied to progerin and, if so, whether progerin’s ability to bind with other progerin molecules could be assessed via this system.
To achieve this, we started by cloning the progerin sequence into our plasmids via Infusion cloning, which in this case required a 2:1 ratio of insert to plasmid (100 ng of insert, 50 ng of plasmid), 2 µL of Infusion solution, and water to bring the final volume to 10 µL. The Infusion reaction was carried out at 55°C for 15 min.
Afterwards, E. coli Top10 cells were transformed with the cloning products and plated on LB Agar + ampicillin/kanamicine plates, which were incubated overnight at 37°C.
The next day, plasmid DNA was extracted via extraction procedure. Before proceeding with yeast transformation, the plasmids were digested with restriction enzymes and analyzed by gel electrophoresis to verify fragments lengths. Specifically, EcoRV and BamHI were used on pGBK, and EcoRV on pGAD, yielding bands of the expected sizes.
S. cerevisiae Y190 transformed for Y2H
Afterwards, we proceeded with yeast transformation. For our work concerning Y2H, we used the S. cerevisiae Y190 strain (different from the CEN.PK strain used for phenotype characterisation). This strain is commonly used in Y2H experiments because it carries specific reporter constructs and genetic features that minimize auto-activation of the reporter genes, allowing more reliable detection of true protein-protein interactions. Transformation was performed using the one-step protocol, as we expected a high transformation efficiency.
At this stage, we encountered an unexpected problem: transformation was successful on only a few plates, and those that showed growth had limited, small colonies. Due to this result, we tested the obtained colonies for progerin expression via Western blot.
Western blot was performed on a liquid culture of the selected clones. Proteins were extracted from 5 OD of culture using the trichloroacetic acid (TCA) protocol. After protein extraction, samples were loaded onto 5% acrylamide SDS-PAGE gel, which was then transferred on PVDF membrane. Ponceau staining confirmed correct protein transfer and migration on the membrane.
Then we started the blotting procedures, which were performed using two different primary antibodies: anti-myc for pGBKT7, and anti-HA for pGADT7, reflecting the epitopes present on the two plasmids.
The secondary antibody was the same for both: anti-mouse peroxydase antibody.
Finally, membranes were developed using Luminol solution and analysed with a Uvitech system.
As we expected from the unusual transformation results we saw on plates, none of the yeast cultures expressed progerin.
We attributed the inability of the cells to grow in the context of progerin expression to the cytotoxic effect of progerin when constitutively expressed. Indeed, the ADH1 promoter which drives protein expression in both pGBKT7 and pGADT7, is a constitutive promoter (unlike the GAL1 promoter of pYES2).
Following this result, we changed our initial strategy regarding progerin expression:
- We decided to express only the C-terminal portion of progerin;
- We chose to express progerin only in pGBKT7
Progerin C-term fragments cloning
The fragments selected for expression, instead of the full-length progerin, were the same as those described in the studies that recognised BUBR1 as a progerin interactor[1]. The regions we chose to express are the following:
- From amino acids 430 to 614 (BBa_25HM35ST): includes the Ig-like domain of the protein;
- From amino acids 545 to 614 (BBa_25IF4C4Z): does not include the Ig-like domain
We chose to continue our experiments using only the pGBKT7 plasmid, as we believed that expression through the BD would limit progerin cytotoxicity, since the BD localizes primarily to its DNA targets and is less likely to diffuse freely within the cell.
Instead of ordering new fragments, we derived our sequence of interest from the pGBK_progerin plasmid already available in the lab and BBa_25NDL8N0, using specifically designed primers:
- In the first procedure, the primer design allowed us to remove the N-terminal region of progerin that we did not want to express (aa 1-430 and 1-545), leaving the C-terminal fragments intact;
- In the second procedure, primers were designed to amplify the fragments of interest by PCR
The cloning results were verified by restriction enzyme digestion and gel electrophoresis, confirming the correct length of our fragments. The enzymes used were EcoRI and BamHI. Finally, we also verified the sequences by Sanger sequencing.
Progerin C-term fragment transformation in S. cerevisiae Y190
Using the C-terminal fragments of progerin, we were able to achieve consistent yeast growth. On top of that, progerin expression was confirmed by Western blot, performed using the same procedure as before.
Based on these results, we concluded that we were now able to use these cultures for the Y2H assay.
BUBR1 N-terminal cloning
When establishing the Y2H system for a new protein such as progerin, it is important to first characterize the protein’s known interactions.
For this reason, before testing our bioinformatically predicted interactors (check out our modelling here) and after our negative result testing progerin with itself, we decided to assess progerin’s C-terminal ability to bind a known interactor: BUBR1.
Since we selected pGBKT7 for progerin expression, BUBR1 N-terminal was consequently cloned into pGADT7. Only the N-terminal region of BUBR1 was selected based on previous studies showing its ability to bind the progerin C-terminal region[1 ]. This sequence was ordered from TWIST, and designed with appropriate extremities for Infusion cloning. Since we cloned it in frame after the Y2H activation domain, we weren’t able to add the RFC10 extremities.
The cloning workflow was as follows:
- PCR amplification of the BUBR1 N-terminal fragments received from TWIST in order to increase its concentration;
- Infusion cloning into pGADT7;
- Transformation into E.coli Top10 and plating on LB Agar + ampicillin at 37°C overnight;
- Plasmid extraction from obtained colonies;
- Verification of correct cloning via restriction digestion, gel electrophoresis, and Sanger sequencing
The secondary antibody was, again, anti-mouse peroxydase, that allowed the membrane to be developed on a Uvitech system using Luminol solution.
After assessing BUBR1 expression, we proceeded with co-transformation with pGBK_progerin C-terminal fragments to perform the Y2H assays.
Yeast two hybrid: co-transformation
We performed single and co-transformation to obtain the following cultures for Y2H experiments (including negative controls):
- pGAD_∅
- pGBK_∅
- pGBK_progerin 430
- pGBK_progerin 545;
- pGAD_BUBR1;
- pGAD_∅ + pGBK_∅
- pGAD_∅ + pGBK_progerin 430;
- pGAD_∅ + pGBK_progerin 545;
- pGAD_BUBR1 + pGBK_∅
- pGAD_BUBR1 + pGBK_progerin 430;
Y2H - progerin interaction confirmation by spot test assay
The final step to assess progerin interaction between progerin and BUBR1 was the spot test on selective medium (SD Glucose -Leu -Trp -His +3-AT), compared with growth of the same cultures on non-selective medium (SD Glucose -Leu -Trp). If the proteins interact, growth on the selective medium should be similar to, or at least approach, the growth observed on the non-selective medium.
These are the results we obtained from the spot test designed to assess the interaction between BUBR1 and the C-terminal fragments of progerin.
| Posizione | Plate 1 | Plate 2 |
|---|---|---|
| 1 | CTR (+) | CTR (+) |
| 2 | CTR (-) | CTR (-) |
| 3 | pGBK_∅ + pGAD_BUBR1 #1 | pGBK_∅ + pGAD_BUBR1 #1 |
| 4 | pGBK_∅ + pGAD_BUBR1 #2 | pGBK_progerin 430 + pGAD_∅ #1 |
| 5 | pGBK_progerin 430 + pGAD_∅ #1 | pGBK_progerin 430 + pGAD_BUBR1 #1 |
| 6 | pGBK_progerin 430 + pGAD_∅ #2 | pGBK_progerin 430 + pGAD_BUBR1 #3 |
| 7 | pGBK_progerin 430 + pGAD_BUBR1 #1 | pGBK_progerin 430 + pGAD_BUBR1 #4 |
| 8 | pGBK_progerin 430 + pGAD_BUBR1 #2 | pGBK_progerin 430 + pGAD_BUBR1 #5 |
The results show that no interaction occurs, at least in this system.
To analyse our bioinformatically predicted interactions using the Yeast Two Hybrid system and confirm their functionality, we selected a small subset from the total pool of our predicted interactors.
Infusion cloning
For this reason, we ordered yeast codon-optimised sequences for the chosen interactors:
- BBa_25R6U8N1: LOGO, optimised for S. cerevisiae;
- BBa_25RLZ3X4: n80_02, optimised for S. cerevisiae;
- BBa_25G9QB6S: Rank_15, optimised for S. cerevisiae;
- BBa_25UZI9YM: Rank_21, optimised for S. cerevisiae;
- BBa_256U2NU6: 62aa11_1, optimised for S. cerevisiae
Once the sequences from IDT arrived, they were cloned into the pGADT7 plasmid (“prey” plasmid, expressing the activation domain “AD”) using Infusion cloning for 15 minutes at 55°C. The reaction was performed at a molar ratio of 1:2 (vector:insert), taking into account the lengths and concentration of the linear plasmid and the interactor insert. Since the interactors are ~50 bp inserts and the plasmid <10 kb, a vector-to-insert ratio of 2:1 was used, as recommended by the In-Fusion protocol.
Bacterial transformation and plasmid extraction
5µl of the plasmid (pGAD + interactor X, where X = 1-5) were transformed into Top10 Competent E. coli cells to obtain colonies for plasmid extraction. The constructs carry ampicillin resistance, so the transformation mix was plated on LB agar + ampicillin and incubated overnight at 37°C.
From successfully grown colonies, five of them per construct were isolated and grown in 5 mL of LB medium with ampicillin at 37°C in agitation overnight. Plasmid extraction was then performed using a miniprep protocol (without a kit) the following day..
The extracted plasmid DNA was quantified using Nanodrop, and 1000 ng of each sample were analyzed by 1% agarose gel electrophoresis. Two controls were included: pGAD_empty and pGAD_BUBR1. Prior to gel analysis, plasmids were digested for 2 hours with HindIII and SmaI, enzymes that each cut the plasmid at a single site: HindIII recognizes AAGCTT and generates 5′ overhangs (sticky ends); SmaI recognizes CCCGGG and produces blunt ends
Expected fragment sizes were:
- pGAD_empty: 520bp and 280bp;
- pGAD_interactor: 950bp;
- pGAD_BUBR1: 1350bp and 700bp
Additional plasmid DNA was obtained by re-inoculating the bacteria in LB + Amp and incubating overnight at 37°C, followed by mini-prep extraction using a kit. This material served both as a working plasmid for experiments and as a stock for the first three interactors in pGADT7, after Sanger sequencing confirmed their sequences.
Transformation in Y190 for Y2H
As we obtained the results from all of the verification analyses, we proceeded to the chemical transformation in S. cerevisiae Y190 to generate the strains required for the Y2H assay.
All sequenced interactors were transformed into Y190 using our “One-step protocol:
- interactors 1,2, and 3: 20 µL and 80 µL of transformation mix plated;
- interactors 4 and 5: 50 µL of transformation mix plated
Three days later, five colonies per interactor were re-streaked and incubated at 28°C for an additional three days. Once sufficient material was obtained, the yeast cultures were co-transformed via chemical method with:
- pGBK_empty and pGBK_progerin545 for all the five interactors;
- pGBK_progerin430 just for the first three interactors
Performing the Y2H assay
At this stage, all constructs were ready for testing, and positive and negative controls were preserved at 4°C. Cultures were inoculated in SD Glu -LEU -TRP medium overnight. The following day, we performed the spot test according to our protocol on both the permissive medium (SD Glu Agar -TRP -LEU) and the selective medium (SD Glu Agar -TRP -LEU -HIS + 3-AT 30 mM), which allows growth only of yeast cells in which an interaction between progerin and the tested interactors occurs.
No growth was observed on the selective medium, indicating that the C-terminal fragments of progerin tested do not interact with the selected partners under these conditions.
| No. | PLATE A | PLATE B | PLATE C |
|---|---|---|---|
| 1 | pGBK_progerin 430 + pGAD_∅ | pGBK_progerin 430 + pGAD_∅ | pGBK_progerin 430 + pGAD_∅ |
| 2 | pGBK_progerin 430 + pool pGAD_interactor 1 #1 | pGBK_progerin 430 + pool pGAD_interactor 2 #1 | pGBK_∅ + pool pGAD_interactor 3 |
| 3 | pGBK_progerin 430 + pool pGAD_interactor 1 #2&3 | pGBK_progerin 430 + pool pGAD_interactor 2 #2&3 | pGBK_progerin 430 + pool pGAD_interactor 3 #1 |
| 4 | pGBK_progerin 430 + pool pGAD_interactor 1 #4&5 | pGBK_progerin 430 + pool pGAD_interactor 2 #4&5 | pGBK_progerin 430 + pool pGAD_interactor 3 #2&3 |
| 5 | pGBK_∅ + pool pGAD_interactor 1 | pGBK_∅ + pool pGAD_interactor 2 | pGBK_progerin 430 + pool pGAD_interactor 3 #4&5 |
| No. | PLATE D | PLATE E | PLATE F |
|---|---|---|---|
| 1 | pGBK_progerin 545 + pGAD_∅ | pGBK_progerin 545 + pGAD_∅ | pGBK_progerin 545 + pGAD_∅ |
| 2 | pGBK_progerin 545 + pool pGAD_interactor 1 #1 | pGBK_∅ + pool pGAD_interactor 2 | pGBK_∅ + pool pGAD_interactor 3 |
| 3 | pGBK_progerin 545 + pool pGAD_interactor 1 #2&3 | pGBK_progerin 545 + pool pGAD_interactor 2 #1 | pGBK_progerin 545 + pool pGAD_interactor 3 #1 |
| 4 | pGBK_progerin 545 + pool pGAD_interactor 1 #4&5 | pGBK_progerin 545 + pool pGAD_interactor 2 #2&3 | pGBK_progerin 545 + pool pGAD_interactor 3 #2&3 |
| 5 | pGBK_∅ + pool pGAD_interactor 1 | pGBK_progerin 545 + pool pGAD_interactor 2 #4&5 | pGBK_progerin 545 + pool pGAD_interactor 3 #4&5 |
| No. | PLATE G | PLATE H |
|---|---|---|
| 1 | pGBK_∅ + pGAD_interactor 4 | pGBK_∅ + pGAD_interactor 5 |
| 2 | pGBK_progerin 545 + pGAD_∅ | pGBK_progerin 545 + pGAD_∅ |
| 3 | pGBK_progerin 545 + pGAD_interactor 4 #1&2 | pGBK_progerin 545 + pGAD_interactor 4 #1&2 |
| 4 | pGBK_progerin 545 + pGAD_interactor 4 #3&4&5 | pGBK_progerin 545 + pGAD_interactor 4 #3&4&5 |
Validation of the interactors against lamin A
To further investigate the specificity of our selected interactors against progerin, the five chosen interactors were tested against its wild-type counterpart, lamin A.
We started by cloning the C-terminal sequence of lamin A (aa 430-646) into pGBKT7 (“bait” plasmid, expressing the binding domain “BD”) using Infusion cloning for 15 minutes at 55°C. The reaction was carried out at a molar ratio of 1:1, taking into account the concentration and length of both the plasmid and insert (pGBK vector: 7200 bp, 70 ng/µl; lamin A BBa_25K3P964: 686bp, 25 ng/µl).
The resulting construct was then transformed in E. coli Top10, and the transformation mix was plated on LB Agar + kanamycin (the vector carries kanamycin resistance) and incubated overnight at 37°C.
The following day, six colonies were selected and inoculated in LB + Kan to perform the mini-prep without kit the next day. The extracted plasmid DNA was quantified using the Nanodrop, and 1000 ng were digested to verify the insertion of lamin A in pGBK.
Plasmids were digested for 2 hours with PstI and EcoRV, enzymes that each cut the plasmid at a single site:- PstI recognizes CTGCAG and generates 3′ overhangs (sticky ends);
- EcoRV recognizes GATATC and produces blunt ends
Digestion was verified by 1% agarose gel electrophoresis, using pGBK_empty as a control. Once correct insertion was confirmed, the pGBK_laminA plasmid was transformed into S. cerevisiae Y190 and co-transformed with the following pGADT7 plasmids:
- pGBK_LaminA + pGAD_∅
- pGBK_LaminA + pGAD_interactor1;
- pGBK_LaminA + pGAD_interactor4;
- pGBK_LaminA + pGAD_interactor5
Transformation and co-transformation mixes were plated on permissive medium (SD glucose -LEU) and incubated at 28°C for growth.
Three days later, five colonies for each interactor were picked and inoculated overnight at 28°C. Positive and negative controls, stored at 4°C, were also inoculated in SD Glu -LEU -TRP overnight.
Once we obtained sufficient material, we performed the spot test, according to our protocol, on both the permissive medium (SD Glu Agar -TRP -LEU) and the selective medium (SD Glu Agar -TRP -LEU -HIS + 3-AT 30 mM), which allows growth only of yeast cells in which an interaction between progerin and the tested interactors occurs.
No growth was observed on the selective medium, suggesting that the C-terminal fragment of lamin A does not interact with the tested partners. This result confirms that our selected interactors do not target lamin A, validating their specificity for progerin.
| No. | PLATE I | PLATE L | PLATE M |
|---|---|---|---|
| 1 | pGBK_∅ + pGAD_interactor 1 | pGBK_∅ + pGAD_interactor 4 | pGBK_∅ + pGAD_interactor 5 |
| 2 | pGBK_laminA + pGAD_∅ | pGBK_laminA + pGAD_∅ | pGBK_laminA + pGAD_∅ |
| 3 | pGBK_laminA + pGAD_interactor 1 #1&2 | pGBK_laminA + pGAD_interactor 4 #1&2 | pGBK_laminA + pGAD_interactor 5 #1&2 |
| 4 | pGBK_laminA + pGAD_interactor 1 #3&4&5 | pGBK_laminA + pGAD_interactor 4 #3&4&5 | pGBK_laminA + pGAD_interactor 5 #3&4&5 |
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- [1] Zhang, N., Hu, Q., Sui, T., Fu, L., Zhang, X., Wang, Y., Zhu, X., Huang, B., Lu, J., Li, Z., & Zhang, Y. (2023). Unique progerin C-terminal peptide ameliorates Hutchinson-Gilford progeria syndrome phenotype by rescuing BUBR1. Nature Aging, 3(2), 185–201. https://doi.org/10.1038/s43587-023-00361-w
To validate our constructs and investigate progerin degradation in a relevant biological context, we performed all our cell-based experiments using MRC-5 human fibroblasts. MRC-5 cells are a diploid, non-tumorigenic cell line derived from normal human lung fibroblasts, commonly used as a model for physiological cellular processes such as growth, senescence, and oxidative stress. Their fibroblastic origin makes them a suitable system for studying progerin-associated phenotypes, which primarily affect connective tissue.
All experiments were conducted at the Department of Molecular Medicine (DMM), University of Padua, inside biosafety level-2 (BSL-2) cabinets, ensuring proper containment and sterile handling. Cell Housekeeping methods and protocols are linked:
- Freezing cell cultures: Freezing_Cells_Benchling.pdf
- Handling cell cultures: Handling_Human_Cell_Lines_Benchling.pdf
- Thawing cell cultures: Thawing_Cells_Benchling.pdf
Each transfection was performed using Lipofectamine™ 3000 (lipofectamine3000_protocol.pdf Thermo Fisher Scientific), following the manufacturer’s protocol, to achieve efficient delivery of plasmid constructs into mammalian cells. Because MRC-5 cells are not included in the iGEM’s White List, we submitted a Check-In form (ID: oE95JbCxfaXB7whj844xfP).
To perform experiments in a disease-relevant model, we used primary fibroblast lines derived from Hutchinson–Gilford Progeria Syndrome (HGPS) patients, obtained through a scientific collaboration with Dr. Giovanna Lattanzi, a leading expert in laminopathies and Head of the Laminopathies and Muscle Biobank at the CNR Institute of Molecular Genetics in Bologna, Italy. These patient-derived fibroblasts faithfully reproduce the molecular and morphological hallmarks of Progeria, including aberrant nuclear morphology, increased oxidative stress, and reduced proliferative capacity, providing an ideal model to test the degradation system under pathological conditions. Since these cell lines are not included in iGEM’s White List, we submitted a Check-In form (ID: f8GE5FZ1Qtx1A9267ZKPS8) to ensure compliance with iGEM safety standards.
As specified in the forms, our cell lines were tested to confirm the absence of mycoplasma and viral contaminants. Official documentation certifying these results, signed by Prof. Claudia Del Vecchio (Microbiology, University of Padua) and Prof. Livio Trainotti (Project PI), is available at the following (Declaration_virus-free_Padua.pdf )
Progeria is often associated with increased oxidative stress and elevated ROS production, contributing to the disease phenotype. To establish this phenotype and test for a potential reversal toward a healthy phenotype, we developed a ROS production assay to assess ROS levels in different transfected conditions.
Experimental Setup:
The experiment was performed in a 24-well plate, 5 × 104 MRC-5 fibroblasts were seeded on Day 0 and transfected using Lipofectamine™ 3000 on Day 1. The ROS Assay Reagent was added on Day 4, and the results were analyzed using the Varioskan™ plate reader on Day 6. The assay was performed in biological triplicates to ensure reproducibility. The assay was performed using Total Reactive Oxygen Species (ROS) Assay Kit 520 nm (Thermo Fisher Scientific) (Total_ROS_Protocol.pdf), following the manufacturer's protocol.
The following constructs were transfected into the wells:
- HA-progerin: To evaluate ROS production due to the presence of progerin;
- SpyTag-progerin: To determine the effect of progerin tagged with SpyTag on ROS levels;
- mEGFP-progerin: A fusion construct of progerin with mEGFP, used to investigate whether the addition of a fluorescent tag alters ROS production;
- RING-mEGFP: To assess the effect of the RING domain (fused to mEGFP) on ROS production, testing whether the RING domain influences oxidative stress;
- RING-SpyCatcher: To analyze whether the combination of the RING domain with SpyCatcher modifies ROS production, potentially due to the structural impact of SpyCatcher;
- mEGFP-NLS: Used as a control to evaluate whether the mEGFP tag alone contributes to ROS production;
- mEGFP-SpyCatcher: A control to test the effect of the SpyCatcher tag in the context of mEGFP-tagged constructs;
- LMNA-SpyTag: A lamin A control construct to compare progerin-induced ROS with a non-pathological lamin protein;
- Negative control: Untransfected cells used to establish baseline ROS production, representing normal ROS levels in cells not subjected to transfection or overexpression.
Reasoning Behind Controls:
The negative control (untransfected cells) was used to establish baseline ROS production, representing normal cellular ROS levels in the absence of transfected constructs.
The HA-progerin construct was used to assess the ROS production induced by progerin, which is known to contribute to oxidative stress. SpyTag-progerin was included to isolate the effect of the SpyTag itself on ROS production, helping us understand whether the tag exacerbates oxidative stress independently of progerin. The mEGFP-progerin fusion construct was employed to examine whether the addition of the fluorescent tag alters ROS levels, as mEGFP tags can sometimes cause misfolding or mislocalization of the protein, potentially influencing ROS production.
The RING-mEGFP construct was used to assess the effect of the RING domain (without the SpyCatcher system) on ROS production. This allows us to determine whether the RING domain alone influences oxidative stress. In contrast, the RING-SpyCatcher construct was used to explore whether the addition of SpyCatcher influences ROS production when combined with RING. This test is essential to evaluate whether SpyCatcher’s structural role modifies ROS production, potentially enhancing or mitigating oxidative stress compared to RING alone. The mEGFP-NLS and mEGFP-SpyCatcher constructs serve as controls to determine whether the mEGFP tag or the SpyCatcher system alone influences ROS production, ensuring that observed changes are due to the specific interactions of progerin and the RING-SpyCatcher system.
Finally, the LMNA-SpyTag control serves as a comparison to progerin-induced ROS production, providing a baseline of ROS levels associated with lamin A (a non-pathological form) and allowing us to distinguish between ROS production due to progerin expression and ROS generated by other factors.
Readout:
By comparing ROS levels across these constructs, we aimed to confirm whether the expression of progerin and its degradation system affected oxidative stress and to assess the potential therapeutic effects of the RING-SpyCatcher system in reducing ROS production.
Cellular viability is a critical factor in understanding the effects of progerin expression and the potential therapeutic effects of the RING-SpyCatcher system. In this experiment, we measured cell viability using the alamarBlue® assay, which provides a reliable readout of metabolic activity and cell proliferation.
Experimental Setup:
The experiment was performed in two identical 96-well plates, 1 × 104 MRC-5 fibroblasts were seeded on Day 0 and transfected using Lipofectamine™ 3000 on Day 1. A second transfection was performed on Day 2, and cell viability was assessed on Day 5 and on Day 7. Fluorescence was measured using the Varioskan™ plate reader. The assay was performed in octuplicate to ensure robust results and statistical reliability. The assay was performed using alamarBlue® Cell Viability Reagent (Invitrogen™) (AlamarBlue_protocol.pdf ), following the manufacturer’s protocol.
The following constructs were transfected into the wells:
- Control: Untransfected cells, serving as a baseline for cell viability;
- Progerin-SpyTag: To assess the impact of progerin expression tagged with SpyTag on cell viability;
- SpyTag-mEGFP-SpyTag + RING-SpyCatcher: To evaluate whether the degradative activity of RING towards mEGFP affects cell viability;
- Progerin-SpyTag + RING-SpyCatcher: To investigate whether Progerin degradation via the RING-SpyCatcher system restores cell viability to normal levels or improves it;
- LMNA-SpyTag: A lamin A control construct to compare the effects of progerin-induced viability defects with a non-pathological lamin protein.
Reasoning Behind Controls:
The Control group (untransfected cells) served as the baseline for cell viability, allowing us to evaluate the normal metabolic activity and proliferation of untreated cells. The progerin-SpyTag construct was included to determine whether progerin expression tagged with SpyTag alone has any effect on cell viability, as progerin is known to induce cellular defects in Progeria.
The SpyTag-mEGFP-SpyTag + RING-SpyCatcher construct was included to specifically test whether the degradative activity of RING towards mEGFP influences cell viability. Since the RING domain has been shown to promote protein degradation, we aimed to determine if this activity, when directed at mEGFP, could affect cell survival or metabolic activity.
The progerin-SpyTag + RING-SpyCatcher construct was included to evaluate whether progerin degradation via the RING-SpyCatcher system restores cell viability to normal levels or improves it. This was key to testing whether the degradation of progerin could reverse or mitigate the cellular defects caused by its accumulation, leading to improved cell survival and proliferation.
The LMNA-SpyTag control was used as a non-pathological comparison to progerin, allowing us to distinguish the specific impact of progerin on cell viability from that of the non-pathological lamin A.
Readout:
By comparing cell viability across these different constructs, we aimed to confirm whether progerin expression and its degradation system affected cell survival and to assess whether the RING-SpyCatcher system could restore or improve viability defects caused by progerin.
Flow cytometry is a powerful technique for analyzing protein expression and degradation at the single-cell level. In this experiment, we used FACS to test whether RING-SpyCatcher could degrade mEGFP in the presence of SpyTag, by evaluating changes in mEGFP expression levels.
Experimental Setup:
The experiment was performed in a 12-well plate. 2.5 × 105 MRC-5 fibroblasts was seeded on Day 0 and transfected using Lipofectamine™ 3000 on Day 1, ensuring uniform cell density. A second transfection was performed on Day 2 with the relevant constructs, and cell analysis was performed on Day 4. The assay was conducted in duplicate for each sample, with a negative control group used to establish the baseline fluorescence for untransfected cells.
On Day 4, we harvested the cells for flow cytometry analysis. Each well was washed twice with PBS to remove residual medium, and cells were detached by adding trypsin and incubating for 5 minutes at 37 °C. Trypsinization was stopped by adding DMEM supplemented with FBS, and the resulting cell suspensions were transferred into FACS tubes. The duplicate wells were transferred into the same tubes. Samples were centrifuged for 8 minutes at 1500 rpm, the supernatant was discarded, and the pellets were resuspended in PBS. This washing step was repeated twice under the same conditions. After the final centrifugation, the supernatant was removed and the cells were resuspended in 250 µL of PBS for immediate FACS acquisition.
The following constructs were transfected into the wells:
- Control: Untransfected cells, used to establish the baseline fluorescence (without mEGFP expression).
- SpyTag-GFP-SpyTag: To evaluate the expression of mEGFP in cells with SpyTag tags, without any degradation system.
- RING-SpyCatcher + SpyTag-mEGFP-SpyTag: To assess the effect of RING-SpyCatcher on mEGFP expression and test whether RING degrades mEGFP in the presence of the SpyTag/SpyCatcher system.
Reasoning Behind Controls:
The Control group (untransfected cells) served as the baseline for fluorescence, allowing us to measure any background signal from cells that do not express mEGFP.
The SpyTag-mEGFP-SpyTag construct was used to measure baseline mEGFP expression levels in cells transfected with the mEGFP construct tagged with SpyTag, but without the RING-SpyCatcher system, to establish normal mEGFP expression.
The RING-SpyCatcher + SpyTag-mEGFP-SpyTag construct was included to evaluate whether the RING domain in combination with SpyCatcher induces mEGFP degradation. A reduction in mEGFP fluorescence would indicate that the RING-SpyCatcher system is effectively promoting mEGFP degradation, confirming the system's functionality.
The negative control (untransfected cells) was included to measure the background fluorescence (or absence of mEGFP) and ensure that observed changes in fluorescence were due to the transfected constructs and not due to non-specific fluorescence.
Readout:
By analyzing the mEGFP signal through FACS, we aimed to assess whether RING-SpyCatcher can degrade mEGFP and to evaluate the effectiveness of the SpyTag/SpyCatcher system in inducing protein degradation.
Western blotting was performed to confirm the expression of progerin and to assess the degradation of progerin-Tag when co-transfected with RING-Catcher. For HA-progerin, we used an anti-HA antibody, while for all other constructs, we used anti-progerin antibody to detect progerin expression, kindly provided by Diatheva as part of our collaboration.
Experimental Setup:
The experiment was conducted in a 24-well plate, 1 × 105 MRC-5 fibroblasts were seeded on Day 0 and transfected using Lipofectamine™ 3000 on Day 1 and Day 2, only for certain wells. Cells were harvested on Day 3 for single transfections, and on Day 4 for double transfections, and protein was extracted for Western blot analysis.
The following constructs were transfected into the wells:
Single Transfection (Day 1, Analyzed on Day 3):
- Control: Untransfected cells, used to assess non-specific binding and background signals;
- HA-progerin: To confirm the expression of progerin tagged with the HA epitope using anti-HA antibody;
- Progerin-SpyTag: To assess the expression of progerin tagged with SpyTag using anti-progerin antibody;
- Progerin-mEGFP: To evaluate the expression change of progerin fused with mEGFP using anti-progerin antibody;
- RING-Catcher: This control helps ensure the antibody's specificity in detecting only progerin in the double transfection with RING-SpyCatcher construct;
- mEGFP-NLS: This control helps ensure the antibody's specificity in detecting only progerin in the double transfection with mEGFP-NLS construct.
Double Transfection (Day 1 + Day 2, Analyzed on Day 4):
- Progerin-Tag + mEGFP: To serve as a baseline for progerin expression, allowing us to compare the effects of RING-Catcher on progerin levels and degradation and it also ensures the same transfection conditions for both samples, without affecting progerin expression;
- Progerin-Tag + RING-Catcher: To evaluate progerin degradation when progerin-Tag is co-transfected with RING-Catcher using anti-progerin antibody. This condition tests whether the RING-SpyCatcher system induces progerin degradation, with progerin-Tag + mEGFP serving as the baseline control for the double transfection setup.
Readout:
By performing Western blot analysis on both single transfection (on Day 1, analyzed on Day 3) and double transfection (on Day 1 + Day 2, analyzed on Day 4) setups, we aimed to:
- Confirm the expression of progerin in the various tagged constructs (HA-progerin, progerin-SpyTag, progerin-mEGFP, etc.);
- Evaluate whether the RING-SpyCatcher system effectively induces progerin degradation, which is expected to reduce the progerin signal in the progerin-Tag + RING-Catcher condition compared to progerin-Tag + GFP
Fluorescence microscopy was used to assess the localization of the RING-mEGFP, progerin-mEGFP, progerin-SpyTag + mEGFP-SpyCatcher, mEGFP-NLS, and SpyTag-mEGFP-SpyTag constructs within the transfected cells. The analysis aimed to visualize the expression and subcellular localization of these constructs, which is crucial for understanding the behavior of the tagged proteins in relation to progerin and RING domains. Fluorescence was analyzed using an excitation wavelength of 488 nm and an emission wavelength of 525 nm, detected in the FITC channel.
Experimental Setup:
The cells were transfected with the following constructs:
- RING-mEGFP: This construct was used to assess the localization of the RING domain tagged with mEGFP. It was important to confirm that the RING domain was properly expressed and localized in the cells, particularly in the nucleus, where it might interact with progerin or other substrates for degradation;
- Progerin-mEGFP: The progerin-mEGFP fusion was used to visualize the localization of progerin in cells. Since progerin has been associated with mislocalization and aggregation, this construct was essential to confirm whether the mEGFP tag interferes with progerin’s natural localization pattern and whether the fusion alters its cellular behavior;
- Progerin-SpyTag + mEGFP-SpyCatcher: This construct was used to assess whether the SpyTag-SpyCatcher interaction affects the localization of progerin. It allowed us to test if the SpyCatcher system alters the distribution of progerin in cells, particularly in the context of protein aggregation or mislocalization, which could be a relevant factor in Progeria;
- mEGFP-NLS: This construct serves as a nuclear localization control, ensuring that mEGFP can be directed to the nucleus when tagged with an NLS. This control helped to confirm that the mEGFP tag itself does not cause mislocalization, providing a baseline for comparison with the other constructs;
- SpyTag-mEGFP-SpyTag: This construct serves to confirm the nuclear localization of mEGFP even if tagged with the double SpyTag. This control helped to confirm that the SpyTag does not cause mislocalization, providing a baseline for comparison with the other constructs.
Methodology:
The cells for these constructs were not re-transfected specifically for this experiment but instead, images were acquired from transfections performed as part of other experiments. On Day 3 (for single transfections) and Day 4 (for double transfections), fluorescence microscopy was used to capture the images of these constructs under appropriate excitation settings for mEGFP.
Purpose:
The purpose of this fluorescence microscopy analysis was to:
- Visualize the subcellular localization of the RING-mEGFP, Progerin-mEGFP, and progerin-SpyTag + mEGFP-SpyCatcher constructs;
- Determine the impact of mEGFP fusion and SpyCatcher on progerin localization, especially in terms of whether it causes aggregation or mislocalization;
- Verify the proper nuclear localization of the mEGFP-tagged proteins, especially in the mEGFP-NLS construct, to ensure that any mislocalization observed in progerin-mEGFP or progerin-SpyTag + mEGFP-SpyCatcher was not due to mEGFP itself but rather the nature of the fusion.
Conclusion:
This microscopy analysis provided important visual data on the localization of progerin and RING constructs within cells, shedding light on the potential effects of the fusion tags and SpyCatcher interaction on protein behavior and subcellular distribution.
In the following paragraphs, we provide a detailed description of the pipeline we followed to generate the constructs required for future transfections.
| Constructs | Registry ID | Reference |
|---|---|---|
| HA-progerin | BBa 25B01XHB | A |
| SpyTag-progerin | BBa_25IIEVZB | B |
| mEGFP-progerin | BBa_25Y190DU | C |
| RING-mEGFP | BBa_256804GM | D |
| RING-SpyCatcher | BBa_255KDS7V | E |
| mEGFP-NLS | BBa_252QSG7W | F |
| mEGFP-SpyCatcher | BBa_257EBH49 | I |
| LMNA-SpyTag | BBa_2505I756 | K |
| SpyTag-mEGFP-SpyTag | BBa_2507I0A8 | M |
Preliminary experiments
Enzyme Functionality Check
First of all, it was necessary to verify the activity of BamHI-HF and XhoI.
Three digestion reactions and one negative control were prepared, for a total of four tubes containing, respectively:
- 150 ng of pcDNA3.1 plasmid + 0.5 µL BamHI HF + 2 µL restriction buffer + H2O up to a final volume of 20 µL;
- 150 ng of pcDNA3.1 plasmid + 0.5 µL XhoI + 2 µL restriction buffer + H2O up to a final volume of 20 µL;
- 150 ng of pcDNA3.1 plasmid + 0.5 µL BamHI HF + 0.5 µL XhoI + 2 µL restriction buffer + H2O up to a final volume of 20 µL;
- 150 ng of pcDNA3.1 plasmid + H2O up to a final volume of 20 µL
The reactions were incubated at 37°C for 40 minutes in a water bath. At the end, an electrophoresis was performed. The expected bands were observed: a single band for the first three samples, since the enzymes have a single cutting site within the pcDNA3.1 plasmid, and a smear in the fourth lane, corresponding to the uncut plasmid. The latter appears in multiple isoforms, differing in the degree of supercoiling, and therefore does not show a single defined band. The functionality of the enzymes was thus confirmed.
When the plasmid was digested, either with BamHI HF or with XhoI, a single band was observed even though the two sites were not overlapping; therefore, a second band at a lower molecular weight corresponding to the DNA fragment between the two sites should also be visible. This does not occur because the sites are only 60 bp, so the fragment migrates too fast and runs off the gel before detection.
Linearization of pcDNA3.1
Before proceeding with cloning, it was necessary to linearize the pcDNA3.1 plasmid by restriction digestion with BamHI HF and XhoI. The pcDNA3.1 plasmid had previously been amplified through transformation in E. coli DH5α cells and then extracted using the PureLink™ HQ Mini Plasmid Purification Kit (Invitrogen).
From the extracted plasmid DNA, three vials were prepared, each containing:
- 5 µg of plasmid DNA (concentration 230 ng/µL, added volume = 21.7 µL);
- 2 µL BamHI HF;
- 2 µL XhoI;
- 3 µL Buffer (10X)
- H₂O up to a final volume of 30 µL.
The reaction was incubated for 40 minutes in a water bath at 37°C. After 40 minutes, 1 µL of BamHI HF and 1 µL of XhoI were added, and the vials are left for another 40 minutes in the water bath at 37°C. An electrophoresis was performed to check the linearization: one sample from each vial and one uncut plasmid sample are loaded as a control.
The bands correspond to expectations: each digested plasmid showed a single well-defined band, and a smear for the uncut plasmid because it exists in multiple isoforms that migrate differently depending on the degree of supercoiling. The plasmids were then purified using the QIAquick Gel Extraction Kit (QIAGEN) and quantified with NanoDrop.
| Sample | DNA (ng/µL) | A260/A280 | A260/A230 |
|---|---|---|---|
| pcDNA3.1 1 | 73.8 | 1.85 | 0.18 |
| pcDNA3.1 2 | 69.3 | 1.86 | 0.30 |
| pcDNA3.1 3 | 71.8 | 1.85 | 0.22 |
pJET1.2 cloning
All constructs arrived lyophilized, were rehydrated, and then used for ligation into pJET1.2.
The reactions were set up in the following way:
| Components | Volume (µL) | Note/Concentration |
|---|---|---|
| pJET1.2 | 1 | 50 ng/µL |
| Insert DNA | See Note | Ratio 1:3 (Vector:Insert) |
| Buffer (10x) | 10 | 10x |
| T4-DNA ligase | 1 | |
| nuclease free H2O | 8 | Calculated Volume |
| Final Volume | 20 |
The ligation product was then used to transform E. coli DH5α competent cells, which were plated on LB agar plates containing ampicillin (100 µg/mL) and incubated overnight.
Procedure:
- Thaw competent E. coli DH5α cells on ice;
- Add plasmid DNA to the cells and gently tap the tube to mix;
- Incubate on ice for 30 min;
- Heat shock at 37°C for 3–4 min;
- Immediately return the cells to ice;
- Add 200 µL LB medium (no antibiotic) and incubate at 37°C for 45 min;
- Plate 100 µL of the culture onto LB agar + ampicillin (100 µg/mL). Spread evenly until dry;
- Incubate plates overnight at 37°C;
- The following day, pick isolated colonies and inoculate into tubes containing 3 mL LB + ampicillin (100 µg/mL).
- Plasmid with restriction enzymes: This control was included to confirm complete digestion of the plasmid DNA by both restriction enzymes. In cases of incomplete digestion, the plasmid may remain intact or be cleaved at a single site, thereby generating compatible ends that could be re-circularized by the ligase. Transformation with uncut or re-circularized plasmid DNA results in colony formation;
- Plasmid only: This control was performed to exclude the occurrence of colonies arising from spontaneous plasmid re-circularization or external contamination, thus ensuring that colony growth in experimental reactions reflected specific ligation events.
The first constructs subjected to this process were A, B, and C. To verify the transformation efficiency of the pJET1.2 plasmid, six colonies per plate were selected the following day. Once the high efficiency of transformation into pJET1.2 was confirmed, three colonies were selected for each of the remaining constructs (D, E, F, I, K, M).
From each selected colony, corresponding inocula were prepared and numbered for identification. These were incubated overnight at 37°C with shaking at 150 rpm. The following day, plasmid DNA was extracted using the PureLink™ HQ Mini Plasmid Purification Kit (Invitrogen) and quantified with NanoDrop.
| Sample | DNA (ng/µL) | A260/A280 | A260/A230 |
|---|---|---|---|
| A1 | 24.2 | 2.10 | 2.10 |
| A2 | 31.7 | 1.56 | 1.81 |
| A3 | 53.1 | 1.79 | 1.87 |
| A4 | 85.8 | 1.50 | 1.26 |
| A5 | 50.4 | 1.75 | 1.64 |
| A6 | 45.4 | 1.78 | 1.82 |
| B1 | 114.6 | 1.85 | 2.11 |
| B2 | 23.5 | 1.94 | 2.08 |
| B3 | 56.7 | 1.82 | 1.88 |
| B4 | 85.9 | 1.87 | 2.27 |
| B5 | 88.9 | 1.73 | 1.95 |
| B6 | 58.4 | 1.87 | 2.37 |
| C1 | 51.9 | 1.90 | 2.37 |
| C2 | 75.3 | 1.81 | 1.89 |
| C3 | 104.5 | 1.45 | 1.13 |
| C4 | 61.2 | 1.89 | 2.12 |
| C5 | 74.2 | 1.88 | 2.19 |
| C6 | 55.0 | 1.88 | 2.25 |
| D1 | 64.4 | 1.84 | 1.87 |
| D2 | 46.8 | 1.91 | 2.04 |
| D3 | 207.5 | 1.89 | 2.23 |
| E1 | 60.9 | 1.88 | 2.10 |
| E2 | 49.1 | 1.91 | 2.23 |
| E3 | 44.2 | 1.90 | 2.16 |
| F1 | 67.1 | 1.87 | 2.08 |
| F2 | 4.5 | 1.84 | 2.16 |
| F3 | 62.5 | 1.87 | 2.05 |
| I1 | 54.0 | 1.86 | 2.03 |
| I2 | 91.1 | 1.72 | 1.52 |
| I3 | 24.6 | 1.80 | 2.01 |
| K1 | 73.0 | 1.85 | 2.05 |
| K2 | 86.6 | 1.85 | 2.20 |
| K3 | 70.9 | 1.81 | 2.01 |
| M1 | 121.1 | 1.89 | 2.21 |
| M2 | 100.0 | 1.88 | 2.22 |
| M3 | 107.8 | 1.90 | 2.15 |
Following quantification, a portion of the extracted plasmid DNA was used to perform a restriction digestion with the enzymes BamHI HF and XhoI.
The reactions were set up in the following way:
| Component | Volume (µL) | Note |
|---|---|---|
| DNA | 1.5 | |
| Buffer CutSmarter (10x) | 2 | 10x |
| BamHI HF | 0.5 | Restriction Enzyme |
| XhoI | 0.5 | Restriction Enzyme |
| nuclease free H2O | 15.5 | Calculated Volume |
| Final Volume | 20 |
The restriction product was then analyzed by electrophoresis to assess whether the plasmid contained the desired insert. Positive samples displayed two distinct bands at different molecular weights: one corresponding to the plasmid backbone, and the other, lighter, corresponding to the insert. If the insert band aligned with the expected molecular weight, the sample was considered positive.
The positive samples were: A3, A4, A5, B3, B4, B5, B6, C1, C2, C4, C6, D1, D3, E1, E2, F1, F2, F3, I1, I2, K1, K3, M1, M2, M3.
We chose to proceed with A3, B5, C2, D3, E1, F3, I2, K1, and M1 because these samples displayed higher DNA concentrations as measured by NanoDrop.
pcDNA3.1 cloning
Restriction digestion was performed on all plasmid DNA from positive samples. Since NanoDrop measurements indicated low concentrations, the entire 42 µL of DNA was used instead of the 5 µg specified by the protocol.
The reactions were set up in the following way:
| Component | Volume (µL) | Note |
|---|---|---|
| DNA | 41 | Fixed for enriching 50 µL final volume |
| Buffer CutSmarter (10x) | 5 | 10x |
| BamHI HF | 2 | Restriction Enzyme |
| XhoI | 2 | Restriction Enzyme |
| nuclease free H2O | 0 | Calculated Volume (the sum of the components is 50 µL) |
| Final Volume | 50 | Target Volume |
Digestion products were run on a 1% agarose gel with wide wells, clearly separating the plasmid backbone (higher molecular weight) from the insert (lower molecular weight). Insert bands were excised and purified using the QIAquick Gel Extraction Kit (QIAGEN).
The first three samples were quantified by NanoDrop to determine the volume required for ligation at a 3:1 insert-to-plasmid ratio. Purified inserts were ligated into the previously digested pcDNA3.1 plasmid, and the ligation reaction was incubated overnight.
The reactions were set up in the following way:
| Component | Volume (µL) | Note/Concentration |
|---|---|---|
| pcDNA3.1 | 2 | 70 ng/µL |
| Insert DNA | See Note | Ratio 1:3 (Vector:Insert) |
| Buffer (10x) | 2.5 | 10x |
| T4-DNA ligase | 1 | |
| nuclease free H2O | 19.5 | Calculated Volume |
| Final Volume | 25 |
Two control ligation reactions were prepared:
The following day, chemically competent E. coli DH5α cells were transformed with the ligation products and plated on LB agar with ampicillin. Two vials of competent cells were also transformed with the control ligation reactions. After overnight incubation, all plates showed growth except for the control plate transformed with linearized plasmid without ligase. In contrast, ≈180 colonies appeared on the plate from the first control, due to T4 ligase re-ligating a 69 bp fragment left between the restriction sites, allowing colony growth.
This unintended re-ligation may also have occurred in experimental ligations. To verify, six colonies from each plate were selected, grown overnight at 37 °C with shaking (150 rpm), and plasmid DNA was extracted using the PureLink™ HQ Mini Plasmid Purification Kit (Invitrogen). Restriction digestion with BamHI-HF and XhoI followed by agarose gel electrophoresis confirmed positive clones.
Positive samples displayed two distinct bands: one corresponding to the plasmid backbone and the other at the molecular weight consistent with the expected insert length. The first gel did not allow us to determine which samples were positive, as the molecular weight marker was lost during electrophoresis.
It was necessary to repeat the restriction reaction and load a new gel, from which the following samples were identified as positive: A3.3, B5.1, C2.3, D3.1, D3.4, E1.1, E1.5, F3.1, F3.2, F3.3, F3.4, I2.2, I2.3, K1.1, K1.2, K1.3, K1.5, K1.6, M1.2.
We selected samples A3.3, B5.1, C2.3, D3.1, E1.1, F3.3, I2.2, K1.3, and M1.2 for sequencing based on band quality and intensity.
Quantification of the positive samples selected for sequencing was then performed.
| Sample | DNA (ng/µL) | A260/A280 | A260/A230 |
|---|---|---|---|
| A3.3 | 237.3 | 1.77 | 2.01 |
| B5.1 | 148.2 | 1.75 | 2.01 |
| C2.3 | 373.5 | 1.93 | 2.31 |
| D3.1 | 123.0 | 1.91 | 2.16 |
| E1.1 | 420.0 | 1.90 | 2.21 |
| F3.3 | 610.0 | 1.94 | 2.32 |
| I2.2 | 421.3 | 1.93 | 2.20 |
| K1.3 | 218.0 | 1.93 | 2.36 |
| M1.2 | 539.2 | 1.83 | 2.25 |
Sequencing
In addition to the positive samples, we also decided to send E1.4 and M1.3 for sequencing. Both displayed two bands that, based on their molecular weight, could correspond to two different constructs. This likely indicates contamination, where two different colonies were inoculated into the same tube: half of the bacteria transformed with one construct and half with another.
Before sequencing, the previously extracted DNA was amplified using the Applied Biosystems™ BigDye™ Terminator v3.1 Cycle Sequencing Kit (Fisher Scientific).
We set up 22 PCR reactions, 2 for each sample: one with the CMV forward primer and one with the BGH reverse primer.
The thermocycler was programmed according to the following protocol:
| Step | Temperatura (°C) | Tempo (minuti:secondi) | Cicli (N°) |
|---|---|---|---|
| Initial denaturation | 96.0 | 2:52 | 1 |
| Denaturation | 96.0 | 0:10 | |
| Annealing | 50.0 | 0:05 | 45 |
| Extention | 60.0 | 4:00 | |
| Final step | 4.0 | Hold | 1 |
Before proceeding with automatic Sanger sequencing, the samples were purified from PCR reagents using the OPTIMA DTR™ 96-Well Plate kit (EdgeBio). The obtained sequences were then compared with the expected ones through BLAStn alignments, where our ordered sequence was set as Subject and the sequencing results as Query.