What Does Engineering Success Look Like?
Science and engineering are processes that consist of constant learning and innovation. Successes in engineering are often incremental, as our team found this summer. Yet the small pieces of knowledge that you gather as you cycle through the engineering process accumulates later into larger successes. Thus, the important part of engineering is the process of designing, building, testing, and learning from the results.
The first step of the cycle is designing possible solutions to a problem being presented. After this step, the engineer should have a blueprint laid for what they will build and test moving forward.
In the next step, there should be some kind of physical product built and ready to be tested. In synthetic biology, this is often DNA constructs that are built and later tested.
The third step is testing what has been designed and built; such as doing experiments in the lab. There should also be data gathered so that it can be analyzed and learned from afterwards.
The final step is to analyze the results from the testing phase, and find some takeaways from those results that can be learned from. This new knowledge is used to inform a new design step.
Our Engineering Process
Our team spent a good chunk of our time throughout the iGEM season going through the engineering process from remote settings. We were not able to show a proof-of-concept for our technology because we were only able to get into lab during the end of the season, although we have begun that process. But the lack of lab space did not stop us from having successes throughout our journey.
The engineering of our toehold detection system began by parsing through existing literature to find and learn about RNA toeholds that have been verified to work in E. coli and cell free. After discovering several potential toeholds that had been tested in E. coli, our team modeled expression levels based on the RBS found in the loop of the toeholds. These toeholds and RBSs, optimized for E. coli, were not optimal for expression in B. subtilis. Not only do Bacillus subtilis use different RBSs, but the distance between the RBS and start codon must be different for B. subtilis, with around 7bp between them. With these differences in mind, we re-engineered our toeholds with optimal RBSs and spacing for B. subtilis.
Our original design plan was to target the coding sequence of cjBlue (BBa_K592011), a blue chromoprotein. To create the toehold, we began by using the software developed by the 2017 Chinese University of Hong Kong iGEM team. Using their software, a user can input a target sequence, and it will return optimal target and toehold sequences, with additional estimates for binding affinities. After imputing the sequence for cjBlue, there did not appear to be any good toehold options with high binding affinities. So instead of using cjBlue as a target, our team switched to using the KanR gene. Targeting this gene would allow us to first verify expression of endogenous KanR by plating on kanamycin media to verify when the target is in the cell.
After settling on a toehold design and building the construct, we could then separate testing into two steps: testing the toehold in vivo with an endogenous target, then testing in conjunction with extracellular target uptake. To further verify the functionality of our toehold, we partnered with the 2020 Edinburgh iGEM team. We designed an experiment to be executed in cell free to test if the sequence could detect KanR. The toehold is coupled to sfGFP as a reporter, and targets a 36bp section of the KanR gene. The target will be introduced to the toehold in cell free, and if the toehold functions as intended, fluorescence can be measured with a plate reader.
We plan to test more toeholds targeting various loci of the KanR gene, to determine what the ideal binding patterns are for B. subtilis. We will integrate this knowledge into our toehold software. Additionally, we will test expression levels of toeholds with alternate RBSs and spacing of the start codon to determine what is optimal for B. subtilis RNA toeholds. We will also determine the leakage of our toeholds in B. subtilis, and how to optimize their design and structure to minimize this noise.
The engineering of our recombination detection system began by repurposing tools used for the miniBacillus project, developed by Wenzel and Altenbuchner (2015). Originally, the team ideated a system that would excise a transcription terminator, allowing transcription and translation of a reporter protein once recombination had occurred. Our team then created a model to see how much fluorescence would be visible, relative to the baseline with leakage. We discovered that when using a stronger promoter, fluorescence may be visible, but more leakage gives a higher baseline, making it harder to determine what is background and what is a signal. When using a weaker promoter, fluorescence would be too dim to allow for good functionality. Additionally, due to the rate of recombination, only a small number of cells would even begin to fluoresce in the first place, making the signal even harder to detect. Our solution to these problems was to change the type of readout we were building.
Instead of a colorimetric or fluorescent readout, we designed and engineered a system that would excise the negative selection marker ManP. In the presence of mannose, cells with the ManP cassette cannot survive. By flanking this gene with homology arms to our target sequence, the recombination triggered by the uptake of the target would remove the ManP gene, allowing cells that had encountered the target to survive in the presence of mannose. To test this new idea out, our team returned to modeling to determine the sensitivity of this detection method. We calculated that at least 1000 cells would be needed per test to reliably get colony growth in the presence of the target.
To further understand that rates of uptake and recombination, our team (now with access to lab space) designed and executed experiments to test the effectiveness of recombination into the B. subtilis genome. By using plasmids that integrate into the AmyE loci of the B. subtilis genome, any plasmids that successfully recombine into B. subtilis would fluoresce and gain kanamycin resistance, allowing for easy selection of transformed colonies. Our team was able to establish that inducibly-competent B. subtilis can be reliably transformed at 37°C with less than 100ng of DNA per 1x108 cells. Our next steps were then to vary the conditions at which we tested competence, such as incubation time, temperature, DNA concentration, and agitation. By varying these conditions, we were able to determine that recombination can occur after 1 hour of inducing competence and 1 hour of incubation with the plasmid DNA at 37°C. Recombination can occur with concentrations of DNA upwards of 300ng, and does not require agitation. Under ideal conditions, the rate of recombination is reliable enough for our system to function as intended.
Now that we have proven that certain parts of the homologous recombination system would work, such as accurate signaling and natural competence in different conditions, we are working towards transforming the plasmid with the negative selection marker into our cells and then adding KanR to see if the system works.
The competence of B. subtilis was an integral part of our diagnostic system design. It was essential that we verify the competence of our strains, and figure out how to optimize its induction for our desired use settings. To do this, we began by finding a basic protocol for transforming B. subtilis. This protocol had an incubation period to induce competence of 3 hours, and was performed at 37 degrees in an incubator shaker. After testing and retesting this protocol, we found that only the xylose-inducible strain 1A976 could be transformed in this manner.
To figure out how to induce competence for the mannitol-inducible strain 1A1276, we designed a new protocol for 1A1276. The cells would be incubated for 1 ½ hours before mannitol was added, then incubated for an additional 1 ½ hours after its addition. The mannitol was then removed before the DNA was added. Using this new protocol, we were able to successfully and reliably transform 1A1276.
After establishing a working protocol for both strains, our next step was to attempt to change variables in the protocol to optimize the test for an at home setting. To do this, we tried inducing competence without shaking, at room temperature and 37 degrees, without the addition of the inducing compounds, and for different incubation times. We found that competence could be induced without an incubator shaker after only an hour of incubation at 37 degrees. We were not able to get transformation at room temperature or without the addition of the inducing compound. However, we have been able to establish that competence could be induced using a household appliance like an oven, as shaking is not needed, and can be performed in a third of the traditional incubation time.
We want our system to be able to be competent without the addition of sugar if possible, so we can allow for passive detection. We talk about the potential applications for that on our Applications Page. We are also planning to run experiments testing growth conditions and times at room temperature, also to look for potential passive uptake and detection.
Our Engineering Successes
- Demonstrating High-Efficiency Transformation From Natural Competence
Due to the COVID-19 pandemic, our team’s access to wet lab space was very limited. Because of this, we focused primarily on running experiments that would inform us on how effective a diagnostic system in B. Subtilis could be in an at-home setting. For us, the first step in this process was ensuring that we could reliably induce competence and transform B. subtilis using a simple procedure. We were able to do this in two cell lines, 1A976 and 1A1276, by simply adding sugar to their growth media to induce competence, then transforming them with ~1μg DNA. Both of these cell lines seemed to have very high transformation efficiency using this procedure as we were able to achieve lawn growth of the transformed cells after allowing them to grow overnight.
- Testing Growth and Competence Conditions
The second step for us, was seeing what the limitations of the competence machinery in B. subtilis are. Specifically, we wanted to see what the limitations were surrounding transformation conditions (i.e. temperature, wait times, etc.) and amount of DNA needed for reliable transformation. To look for these limits, we ran a variety of experiments where we varied the temperature the cells were induced at (37C and room temperature), whether they were kept in a shaker, the amount of time given to induce competence (1hr, 2hr, and 3hr) and the amount of DNA added to the cells. From these experiments, we determined that competence can be achieved with less efficiency after only 1 hour of competence induction and ~400ng DNA added, and that reliable transformation can be achieved without the use of a shaker (but incubated at 37C) when ~400ng DNA was added.
- Validating Our Toehold Construct In Vitro
Throughout the competition, we had a very meaningful meaningful collaboration with the iGEM team at Edinburgh. In return for verifying their protocols, they offered to test out our toehold construct in vitro to verify that it works before we try transforming it into our cells to test in vivo. The result seen below shows that our toehold works and we designed it successfully.