Experiments

Here you can find all the experiments we did.

On this page, we have gathered the list of experiments and our goals we have aimed for in our wet-lab work. We divided our wet-lab work into three sections, focusing on ribosome display, bioreporter cloning, and GFP-DARPin expression separately. To see more detailed information about all the labnotes from the experiments check out our labbook and protocols.

Labbooks

Goals of the Experiments

  1. Ribosome Display for DARPin affinity selection
    • Expression of GFP
    • Synthesis of own DARPin library
    • Affinity Selection of DARPins
  2. Bioreporter
    • Bioreporter cloning
    • Bioreporter testing
  3. GFP-binding DARPin synthesis
    • Diffusion of DARPin through a hydrogel

Ribosome Display

Idea

We chose ribosome display as the method for the binding selection, as it has been described as a good method for protein-peptide binding assays (Chen et al. 2021). The idea behind the method is to do in vitro transcription and translation which will result in the targeting peptide being “stuck” on the ribosome. It is crucial that the targeting protein does not have a stop codon and a long enough spacer after the protein sequence, to result in the peptide being far out from the ribosome but still attached to the ribosome, see Figure 1 (Lipovsek and Plückthun, 2004 ).

Ribosome Display
Figure 1. Schematic overview of the ribosome display (created with Biorender.com)

Aim

As we wanted to prove that the selection method could be successful for our AIP binding DARPins, we firstly wanted to prove the method with a known protein-protein binding. We chose GFP binding DARPins as our test targeting protein, and naturally GFP as the target protein. For this, we aimed to produce our own GFP protein by expressing superfold-GFP in E. coli expression cells. We also aimed to test our own AIP binding DARPins, which meant we built a small library of potential AIP binding DARPins. To read more about the background of the selection of DARPin sequences, please visit our modelling page. For the experiments, we aimed to test both our own AIP binding DARPins, as well as our known GFP binding DARPin. To prove the quality of the binding affinity, we also placed our AIP binding DARPin in the same reaction with the GFP binding DARPin. This will prove that the most suitable DARPin will always bind to the specific target.

Testing

We ordered the needed GFP-binding DARPin from IDT. The construction of the fragment can be seen below in Figure 2.

Sequence of GFP-binding DARPin
Figure 2. Ordered GFP-binding DARPin composite part for ribosome display

After we received the DNA fragment, we proceeded with the in vitro transcription and translation according to the protocol.

After in vitro transcription and translation, elution of RNA from the mRNA-ribosome-protein-complex is needed to isolate mRNA from the selection of binders during the ribosome display, for sequencing and identifying, which DARPin bound the best.

The mRNA then needs to be purified and reverse transcribed, for these steps we used ready-made kits, see protocols for the exact methods and tools used.

The last step before sequencing and finding the best suitable DARPin, we needed to prepare the cDNA for sequencing by adding on specific overhangs needed for the sequencing. The forward and reverse overhang can be seen in Figure 3, the same overhangs were added to all DARPin cDNA fragments. After this the cDNA DARPin fragments with correct overhangs were sent for sequencing. We acknowledge DNA Sequencing and Genomics Laboratory, Institute of Biotechnology, University of Helsinki for sequencing.

Sequence of primer overhangs
Figure 3. Overhangs for sequencing

Results

We performed in total three ribosome display experiments. Two of them were affinity selecting our 42 DARPins against the biotinylated AIP1 target, while one of them was affinity selecting our GFP-targeting DARPin against GFP. For the ribosome display containing the GFP-targeting DARPin we added additionally a small aliquot of DARPin #12. This would confirm that the GFP-targeting DARPin would bind the GFP target in higher quantities.

Based on the results from the sequencing, we were successful in the ribosome display with our GFP binding DARPin. The sequence logo for our GFP DARPin and the retrieved sequence from the binding assay can be seen in the Figure 4. It was shown that the sequence found to be bound was actually the GFP DARPin and not the added DARPin #12.

Sequencing logo comparing template GFP-DARPin and results of ribosome display.
Figure 4. Data sequencing logos of the GFP-binding DARPin template (left) and the sequenced results of the ribosome display (right).

From our results in the wet-lab we could demonstrate that sequencing was successful. Specifically, the N-cap for the GFP-targeting DARPin was completely identified, while slight changes of the sequence occurred later. The majority of the sequence presented, however, was the GFP-targeting DARPin. Therefore, we were able to demonstrate that the ribosome display can be used for future experiments, and could be used as an easy tool to find the right DARPin sequence for the target signalling molecule.

We also saw some affinity of sequencing of the AIP binding DARPins. We could retrieve from the sequencing data the same N-cap and beginning of the DARPin sequence. However, unfortunately, due to time restrictions, we couldn't show any specific DARPin that showed great affinity against our AIP. More time for testing and re-runs of the ribosome display would be needed to confirm the exact DARPin sequence that bound the best.

Bioreporter

Idea

The idea behind our bioreporter is to prove the impact that the DARPins have on the signalling molecules. We wanted a bioreporter that was able to produce the needed membrane proteins and internal signalling proteins, to bind the signalling molecules (AIP's) and then give an output to show that the cell has received the information of the binding. For this part of the project we took inspiration from a last year's iGEM team (Team IISER, Kolkata, 2021 iGEM team), who had built a bioreporter with the parts aimed to sense signalling molecules released from S. aureus bacteria. They had successful results, which made us confident that we can build a similar bioreporter but aimed for S. epidermidis.

Aim

We aimed to clone two composite parts that we ordered through IDT for the bioreporter. The first part will have both promoters that we need, one for transcription of GFP, and one for transcription of AgrC and AgrA genes. As proven by the IISER Kolkata iGEM team, it is crucial to have the promoters in opposite directions to avoid any leaking of expression from the strong constitutive promoter. The other composite part will include the needed membrane protein AgrC and the internal signalling protein AgrA. After cloning, we will measure the output signal (GFP fluorescence) when the bioreporter has AIP's in its surroundings, and if possible test the bioreporter when both AIP's and our AIP binding DARPin are in the surroundings of the cell.

Testing

The two composite parts needed for the bioreporter were ordered directly from IDT. The final used and modified parts can be seen in Figure 5.

Sequences of part 1 and part 2 of the bioreporter
Figure 5. PART1 and PART2 ordered for bioreporter

Both parts were amplified and checked with gel electrophoresis. We started with PART2 cloning as that part arrived before PART1 from IDT. This was due to changes that needed to be made, to read more about the changes, see our engineering page.

Both parts were digested with the correct restriction enzymes to get the right restriction sites and were then ligated and transformed into BL21 E. coli expression cells according to protocols. The set up of the plasmid and restriction sites can be seen in Figure 6.

Schematic circular overview of bioreporter.
Figure 6. Bioreporter construction

To test the bioreporter, our aim was to test the GFP intensity of the bioreporter cells when they were induced with AIP's, and then if we would have time, test if the signal would be lower in case DARPins were present in the environment. Unfortunately, we only had time to test the bioreporter with AIP inducement.

For the testing we wanted to know if different concentrations of AIP's would affect the GFP signal, or if it would be the same as long as the threshold of inducement had been achieved. The exact amount of AIP molecules needed to activate the P2 promoter has not been measured as far as we know in literature. However, we had done some estimations of the needed concentration for our modelling work, and for this we chose to see if high concentration would yield high intensity. We ordered the AIP's directly from Genscript as purified protein. The AIP's were dissolvable in DMSO, however, we ran into some trouble of not all protein dissolving in the DMSO when we were preparing the dilutions. Due to this we had only some type of estimates of the concentrations but renamed them to high and low concentrations of the AIP's. The lower concentration should be somewhere around 250nM, however, this is unfortunately not tested due to a lack of equipment available. The amount of each tested AIP's can be seen below in Table 1.

Table 1. Amount of AIP for bioreporter inducement
High concentrated Low concentrated
5 µL 50 µL
2 µL 20 µL
1 µL 10 µL
5 µL
2 µL

The cells were grown up to 1.2 OD600, we added 5uL of culture to 200uL of LB media, which equals 4.8*106 cells in the beginning. After that we induced 3 wells with each concentration and measured the OD600 and GFP intensity at both 470 and 511nm wavelength. The measurements were done with the BioTek Synergy H1 Plate Reader, and the measurements were taken for 10 hour every 20 min.

As blank we had LB media with kanamycin to avoid contamination growth, and as negative control, we had the same expression cells as for the bioreporter, but these ones included the GFP DARPin expression cassette. The set up on our 96 well plate can be seen in Table 2.

Table 2. 96 well plate set up for GFP and OD600 measuring Table showing 96 well plate.

Results

We successfully cloned both composite parts into the target plasmid containing our GFP gene. The bioreporter was assumed to be functional after checking by PCR for the reason of performing restriction cloning, which would align the parts in the direct direction.

The final test of the functional bioreporter was also somewhat successful. We received the GFP signal from all bioreporter cells, which had the GFP gene, and no signal from our negative controls which didn't have the GFP gene. We can conclude that some changes should still be done to the setup of the promoters in the bioreporter. It seems that the P2 promoter might be active even if it hasn't been induced, or then the strong constitutive pBAD promoter has something to do with the expression of GFP. However, it seems not likely as it is about 70bp downstream from the GFP encoding gene, and the pBAD promoter is inserted to start transcription in the opposite direction. Future testing of inserting just the P2 promoter in the plasmid prior to the GFP gene would be needed to figure out what might be the cause of the false GFP signal, for those bioreporter cells which gave a GFP signal even thought they were not induced with the AIP molecules, which is needed for activation of transcription of the GFP gene.

GFP DARPin synthesis

Idea

As a part of the proof of concept of our project, we also wanted to prove how we could produce the DARPins in genetically modified organisms. We wanted to express our test GFP binding DARPin also as a part of our partnership with the Dresden iGEM team, who wanted to do testing with our protein from their developed hydrogel.

Aim

Our aim was to clone our composite part which includes the GFP binding DAPRin gene, as well as a His-tag for purification and an Avi-tag for other downstream processing. We aim to transform this modified pET42b-HF-BE3 plasmid with our target genes into E.coli expression plasmids and induce the cells with IPTG, extract the proteins from the cell, and purify the protein.

Testing

We ordered from IDT the needed DNA fragment, including the T7 promoter and RBS, the needed His-tag and Avi-tag, the GFP-binding DARPin and the termination of the gene with a stop codon and additional His-tag. To speed up the process of the cloning we ordered the fragment with the correct needed restriction sites at the end to fit into our cut plasmid. The ordered fragment can be seen in Figure 7.

Sequence of GFP-binding DARPin expression construct.
Figure 7. GFP-binding DARPin expression construct.

After receiving the fragment, we digested both the pET42b-HF-BE3 plasmid and our fragment with XbaI and XhoI restriction enzymes and did ligation and transformation into BL21 E. coli expression cells.

After this we inoculated a few colonies overnight, at different temperatures, and different amounts of time. We then did the main culture, which we induced with IPTG and did the protein extraction by sonication, all according to the protocol. The protein was then purified with Ni-NTA columns to the best ability, however, most of the expressed protein remained in the pellet.

Results

The GFP-targeting DARPin was mainly present in the pellet and not in the soluble fraction. The best results were present for induction of the GFP-targeting DARPin expression at 20 °C overnight. However, expression and extraction could not be improved. Therefore, we decided to proceed with in vitro transcription and translation of the protein due to the time constraint for our collaboration with TU Dresden.

References

  1. Chen, X., Gentili, M., Hacohen, N. and Regev, A. (2021)
    A cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies
    Nature communications, 12(1), pp.1-14
    Google Scholar
  2. Lipovsek, D. and Plückthun, A. (2004)
    In-vitro protein evolution by ribosome display and mRNA display
    Journal of immunological methods, 290(1-2)
    Google Scholar