Aalto-Helsinki iGEM teams over the years have embraced the spirit of community by sharing their experience to help other iGEMers, and this core value of ours is epitomized through the contribution of our alumni teams to iGEM. This year, we are proudly continuing the tradition - we have compiled all our wet lab troubleshooting details here to help out others who may be facing the same issues as we did in the lab. We have been active contributors to the parts registry this year by adding 11 basic parts and two composite parts. Finally, through our collaboration with Team Aboa from Turku, we have cleared the ground for the foundation of new iGEM teams in Finland.
We designed 11 basic parts and two composite parts, and submitted them to the parts registry. The basic parts contain genetic regulatory elements for protein expression in Escherichia coli and Saccharomyces cerevisiae, while the composite parts include a superfolder green fluorescent protein (sfGFP) reporter that can be used to test promoters. More detailed information on the parts can be found in the Parts page.
Wet lab Troubleshooting
Wet lab experiments are never without their own mysterious issues, and we had a fair share of them with our experiments too. A significant number of these issues were tackled through dedicated troubleshooting. Some issues were resolved just by changing the components or altering the reaction conditions, but other issues persisted for a long time and those required our devoted attention. Here, we have compiled all the troubleshooting measures that we employed to sort our experiments.
Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a common molecular biology technique employed in cloning. Due to the involvement of many different variables, PCR can be a reaction that requires extensive troubleshooting. PCR programs might vary depending on the polymerase being used or on the size of the DNA to be amplified. We troubleshooted some of our own PCR problems, and would like to provide some suggestions to improve the PCR setup.
It is highly recommended to make a master mix depending on the number of samples to be set up for PCR. A master mix will prevent pipetting errors and can lead to a more accurate composition of the reaction across multiple tubes. Additionally, it is important to use controls whenever possible - a positive control and a negative control. Usually the positive control is a DNA sequence that is certain to be amplified, while the negative control consists of every reaction component except the template DNA (the negative control is sometimes also referred as “no template control” for this reason).
For starters, it is always recommended to begin testing your primers in a range of annealing temperatures through a gradient PCR, as the calculated Tm might vary slightly depending on the reaction setup. Setting up a range of reactions between Tm ± 2ºC is a good place to begin.
You can find our PCR protocol in the Protocols section on our Wet lab page.
Extraction of DNA from agarose gels was one of the experiments we dreaded performing due to very poor yields, sometimes as low as 10%. We used commercially available spin column-based kits to extract the DNA from the agarose gel. Since our workflow extensively involved cloning, we found ourselves performing gel extraction a considerable amount of times. We tested a variety of different protocols and have optimized our own that improved yield to around 60%. The optimized protocol is as follows:
- Use <1% agarose gels and extract the gel slice, cutting as close to the DNA band as possible.
- Add twice the volume of binding buffer to the gel slice (for example, add 200 μL of binding buffer to a gel weighing 100 mg).
- Melt the agarose in the binding buffer at 50ºC with shaking at 100 rpm until the agarose gel has completely melted. Occasional vortexing helps to melt the gel quickly.
- Load up to 700 μL of the molten agarose with DNA sample to the spin column and centrifuge at 11000 x g for 1 minute. Collect the flow through and add it again to the spin column to increase the amount of DNA bound to the column.
- Repeat the above step to load any remaining sample.
- Add 500 μL of the wash buffer to the spin column and centrifuge at 11000 x g for 1 minute, discard the flowthrough.
- Repeat the wash step one more time and discard the flowthrough.
- Centrifuge the empty spin column at 11000 x g for 2 minutes to collect any remaining ethanol from the wash buffer. Place the spin column with its lid open in a heat block at 50ºC for 2 minutes to evaporate the last traces of ethanol.
- Add 30 μL pre-warmed elution buffer or milliQ water to the center of the column and incubate at room temperature for 2-5 minutes followed by centrifugation at 11000 x g for 1 minute.
- Repeat the elution step with another 30 μL of elution buffer or milliQ water.
Some things to consider when performing the gel extraction:
- Use a freshly-prepared agarose gel to perform gel electrophoresis. Less than 1% is recommended.
- Allow the gel slice to melt completely before loading the sample to the column as any remaining solid agarose could block the membrane in the spin column.
- Binding buffer can also be added to a volume of 2.5x the weight of the gel slice. This is useful for gels of 1% or more.
- Elution with water is recommended unless the extracted fragment is intended for long term storage at -20ºC.
- Note: Improvement in yield might result in reduced concentration when eluting with two x 30μL of elution buffer.
Modular Cloning (MoClo) is a Golden Gate cloning technique that utilizes type IIS restriction enzymes to facilitate the construction of large multigene assemblies in an efficient way. Work with MoClo is straightforward and the reactions are easy to set up in a thermal cycler. We have worked with the MoClo reaction setup suggested by the Boston U iGEM team, and it worked for most of our Level 1 constructions. However, the molar concentrations of the DNA parts involved can be a cause for concern. We had little success using the suggested 10 fmol for each DNA part. The number of successful clones increased when we used 50-100 fmol instead. The number of successful clones also increased when we changed the ratio of vector:insert from 1:1 to 1:3.
Lastly, we also recommend using at least half of the reaction for transforming competent Escherichia coli cells, irrespective of their competency, to increase the chances of obtaining a positive clone.
Our protocol for a standard MoClo reaction can be found in the Protocols section on our Wet lab page.
Future of iGEM in Finland
During our October visit to Turku, we discussed with Team Aboa how we can introduce new teams in Finland. After the Wiki Freeze and simultaneously with our own Aalto-Helsinki iGEM recruitment, we will begin contacting other Finnish universities in an effort to promote new iGEM teams and synthetic biology in general. Read more about this meeting on our Collaboration page here.
Testaments to Help Future Aalto-Helsinki Teams
Since Aalto-Helsinki is comprised of 10 new students every year, we found that we were constantly struggling and asking questions about the logistics, finances and organization of an iGEM team. The Aalto-Helsinki 2020 team has been very helpful in answering our questions and providing their documents. However, at the start of the iGEM project, we were quickly overwhelmed with all the information they provided and found the entire process difficult to navigate. As a result, we have decided to create team testaments about how our team runs and how iGEM is organized in general for next year’s team. The testaments will provide crucial information and tips on how to be an iGEM team leader, what is the responsibility of each team-member, and tips and tricks that we wish we would have known when starting and completing our iGEM project. We have worked on these testaments throughout the summer, and will have them finalized in January in time for the start of the new Aalto-Helsinki 2022 team.