- First PCDA synthesis using different concentrations for proof of concept. We tried to make a stock solution which did not work due to low solubility.
- Preparation of Brain Heart, Infusion, buffers, and solutions.
- Inoculation of C. acnes and preparation of glycerol stocks.
- Buffer preparations.
- LTA-bead conjugation.
- Library preparation (resuspension).
- 1st round of SELEX.
- No results, extensive troubleshooting and reruns.
- Increased amount of PCR cycles, different template concentrations.
- Noticed we had bought the wrong reverse primer, it had biotin on the wrong terminal.
- Another mistake that we noticed was that our protein A was stored at RT instead of the - 35 to - 25 degrees Celcius that is recommended on the package. According to instructors, this was nothing to be concerned about.
- A new PCDA solution was created with a concentration of 1 mM - the same concentration as is required for coupling. This solution became deep blue after polymerisation. This was subsequently used for all conjugation attempts.
- Aptamer coupling with PCDA was tested with different concentrations of PCDA in the coupling process, from a volume of 50 uL PCDA as suggested in the protocol to a maximum volume of 237.5 uL. A higher PCDA concentration seemed to provide a higher yield after dialysis, meaning that more PCDA vesicles were conjugated to the same amount of aptamers.
- Protein A coating of plates for protein A aptamer testing. The aptamer coupled to PCDA was tested in the protA wells for binding and colour change, which did not yield any colour change. Further investigation was needed.
- PCDA testing with heat. Heat was used to test if a colour change is possible: the PCDA solution did indeed turn to a bright orange when subjected to a burner. The PCDA was also tested with UV light, which did induce a colour change, although very slowly. The solution turned to a deep purple when left under UV light overnight.
- Start of cell SELEX using C. acnes as targets.
- Protein A aptamer testing using affinity chromatography. A Mabselect column used for IgG purification was used to test the binding of our protein A aptamer. The aptamer solution ran through the column when the sample was applied, meaning that no binding took place. An attempt was made using magnetic protein A beads, with the same results. It is possible that the aptamer binds to a different part of protein A than is accessible in the column and on the beads.
- Finally, since none of the SELEX runs worked, and after extensive troubleshooting using different annealing temperatures, working concentrations and cycle numbers, we had a meeting with our advisor Dimitri. We concluded that the libraries required HPLC purification which we did not choose when ordering. Thus, a new library was ordered with HPLC purification.
- After consulting our supervisors about the PCR protocol, it was pointed out that our template concentration was excessively high in the PCR reaction. The consequence of this is that the DNA strands don't have the time and right conditions to denature for the PCR to work - they are too dense (3 000 ng vs. < 500 ng). Therefore, a PCR was done with "normal" concentrations, obtained from the polymerase's documentation. When reading the documentation, the dNTP concentration was too low (1 uM vs. 200 uM) and the primer concentration was too high (2.5 uM vs. 1 uM).
- Once the PCR was working and amplification bands were visible, we discovered that we had contamination since the NTC lane also displayed a band. Thus, extensive troubleshooting was done to figure the source of the contamination out. Tubes were prepared with one component switched for a new one in each. The gel showed that the dNTP tube was the source of the contamination.
- A new SELEX amplification was performed, yielding no bands. Again, a new troubleshooting step had to be started, testing two different polymerases, two dNTP concentrations, as well as two annealing temperatures. Contamination was again present in the NTCs, so we used new polymerase and buffer, which worked.
- We performed our first working PCR amplification of our SELEX round 1 library, which yielded distinct bands. The next step was to separate the sense and antisense DNA strands, followed by desalting. This step was thought to be not working, but it turned out that the final concentration of aptamers was just very low. A final PCR was therefore done to check if aptamers were present in the end. Again, some troubleshooting was required since we had contamination, this time stemming from the primers.
- The protein A aptamer binding assay was designed for the fluorescein-tagged aptamer and using a plate reader. The first step was to make a calibration curve to see which aptamer concentrations are suitable for detection. After that, the binding assay was performed, showing that the aptamer binds to protein A.
- A new SELEX round was started in order to test our troubleshooting guide and gained experience. This did not work since we got contamination in our PCRs, even after trying again two times with new aliquots of PCR reagents. Due to time constraints, no further attempts were performed and our focus moved on to testing the protein A aptamer and sending transformation samples for sequencing.
- A final protein A aptamer binding affinity test was run with the same method as before, showing no binding to protein A.
BioBrick Engineering Workflow
- Preparation of LB, LB-agar plates and stock solutions.
- The team learnt basic techniques: culturing bacteria in liquid LB and plating in LB-agar, E. coli transformation.
- Cloning was started with the aim of practicing and creating a biobrick with protein A with a GFP attached. A plasmid from the 2019 distribution kit was used but it did not work, although the same plasmid from the 2021 distribution kit worked.
- The cloning strategy was designed: we decided to clone protein A and a reporter protein together in the same vector. AmilGFP (yellow chromoprotein) and GFP (green fluorescent protein) were chosen as potential reporter genes. Protein A, amilGFP and GFP BioBricks were obtained from the 2021 DNA Distribution Kit Plates. We chose several GFP BioBricks in case the transformation efficiency was not high in all of them.
- Protein A: BBa_K103003
- GFP: BBa_K608008, BBa_K608010, BBa_K608011
- AmilGFP: BBa_K1073024
The initial strategy was to digest both BioBricks (protein A and reporter protein) and ligate them together in a new open vector with Ampicillin resistance (PSB1A3).
- Several rounds of transformation, O/N culture, glycerol stock + miniprep were performed.
- After several attempts we got a good plasmid prep and glycerol stocks for each plasmid.
- Digestion with the corresponding restriction enzymes was performed. Ligation mixtures containing of PSB1A3 + Protein A BB + reporter gene GFP(BBa_K608011) or amilGFP (BBa_K1073024) were set. We realised that we did not use Dpnl to degrade the RFP present in vector plasmid PSB1A3.
- Decided to simplify the cloning strategy: It is better if we deliver the construct in PSB1C3 rather than PSB1A3. The new strategy consists in digesting the Protein A BioBrick and then, ligate it in a linearized plasmid containing the reporter gene (GFP or amilGFP).
- Plasmids containing the reporter gene GFP(BBa_K608011) or amilGFP (BBa_K1073024) were digested with SpeI and PstI. Plasmids containing the Protein A BioBrick were digested with XbaI and PstI. Ligation reaction was set afterward.
- In order to be able to transform E. coli with the ligated construct we need to purify it from the gel. The ligation mixtures are not enough to do gel extraction.
- We decided to keep the cloning strategy but made slight changes: we decided to amplify Protein A BioBrick by PCR, using VF2 and VR standard primers. Then, digest the PCR product with EcoRI and XbaI. This digested product will be ligated with a higher amount of linearised plasmids containing the reporter gene GFP(BBa_K608011) or amilGFP (BBa_K1073024). This way we can perform gel extraction after ligation.
- PCR on Protein A BioBrick followed by a gel extraction (PCR clean-up was not an option, very small fragments) were performed.
- Digestion followed by gel extraction were performed in plasmids containing the reporter gene GFP(BBa_K608011) or amilGFP (BBa_K1073024). Optimisation of this step was needed.
- Ligation of Protein A + GFP and Protein A + amilGFP.
- TOP10 E. coli competent cells were transformed with the ligation mixtures. Glycerol stocks and minipreps from 3 colonies of each combination of Protein A + reporter gene were set.
- The plasmid preps were sent for sequencing using VR and VF2 primers. The BioBricks sequences were correct but we realised we need to remove the STOP codons in between protein A CDS and reporter gene CDS.
- Quikchange Site-directed mutagenesis strategy was designed to change the STOP codons (TAA) for Ser (TCA). Protein A + Amil-GFP construct has 2 STOP codons in-between CDSs, but Protein A + GFP has 4 STOP codons and the CDS are not in-frame. Due to the time restriction, we decided to move on only with the Protein A + amilGFP construct. Primers for QuikChange mutagenesis were designed using Kozane.app.
- Quikchange protocol was designed taking into account the Aligent protocol and Phusion polymerase specificities. Since the annealing temperature is tricky to calculate (primers don't bind completely to the target sequence) we performed a gradient PCR using 3 different annealing temperatures: 55, 58 and 60 degrees Celsius.
- TOP10 E. coli competent cells were transformed using different volumes of Quickchange DNA mix: 5, 10 and 15uL.
- Yellow colonies were only in 55 and 58 degrees Celsius plates. 8 colonies from the 55 degrees Celsius plate and 20 from the 58 degrees Celsius plate were used to set O/N cultures and glycerol stocks. Minipreps of 8 O/N cultures from 55 degrees Celsius and 8 O/N cultures from 58 degrees Celsius and the 16 samples were sent for sequencing.
Feel free to read further into what we did in the lab in our lab books
Synthetic Biology Lab Manual
Courtesy of Josefine Liljeruhm, Erik Gullberg and Anthony C. Forster, Uppsala University.
The above protocol is an open access iGEM protocol by Paul Rutten from The University of Oxford et al.
The above protocol is repeated in the Gel Extraction protocol, this is simply the official company card.