Team:Northwestern/Human Practices
HUMAN PRACTICES
Synthetic biology is a field that permeates through various sectors of society. With a scientific goal in mind, it is crucial to take the time and understand how this objective can make changes socially, politically, economically and of course scientifically. It is also important to assess the overarching goal of this research and its future impact. By participating in meetings with experts in the field, market research on existing products, and outreach with local student groups, our team reevaluated our project design to come one step closer to reaching our goal.
EXPERT INTERVIEWS: WHOLE-CELL VS CELL-FREE SENSORS
Our project design and direction was largely informed by interviews we had with experts in the synthetic biology field. One of the biggest considerations we kept in mind this summer was whether our system would be more practical and impactful as a cell-free or whole-cell sensor. We met with cell-free experts to determine if this was something we could potentially incorporate into our biosensor and to learn how users would interact with this type of system. Since our product is dependent on cellular processes, including DNA replication and SOS induction, we wanted advice from experts about if we could create and sustain our biological circuit without cells. We hoped to gain more insight into safety considerations and implications with cell-free systems.
Interview with Dr. Ashty Karim
To better understand how cell-free systems function and how we could potentially incorporate our system into a cell-free sensor, we interviewed Dr. Ashty Karim, Chemical Engineering PhD. Dr. Karim is a research fellow and assistant scientific director in the Jewett lab at Northwestern. His research focuses on natural product discovery and biosynthetic pathway prototyping for cell-free systems.
Dr. Karim explained how if we chose to go cell-free, we could potentially add exogenous genomic DNA to the cell-free lysate. In choosing this exogenous DNA, we would have to do research into which sequence would be the best replacement for the genome. Another important consideration is that we would have to express all proteins involved in our system beyond native levels.
Dr. Karim also went over the benefits and problems with cell-free, as well as the general lab process. He highlighted that although cell-free systems would give us control over protein proportions and less engineering would be involved, the duration of a reaction in a cell-free system and the signal readout sensitivity of paper biosensors pose as limitations. Cell-free paper must also be kept dehydrated and is a bit more expensive than whole-cell systems, but it would be shelf-stable for up to a year.
Takeaway: Ultimately, this meeting gave us insight into how users would interact with a one-time use DNA damage sensor and gave us direction into what information we needed to seek from literature to make a cell-free system plausible. After this meeting, we began to brainstorm how to overcome the issue of genomic DNA being completely removed in lysates and how to improve the visual output of our system.
Interview with Adam Silverman
To clarify which plasmids and proteins we would potentially want to be present if our project was in a cell-free system, we met with Adam Silverman, a Chemical and Biological Engineering PhD student. Adam works extensively with cell-free biosensors.
Adam highlighted that incorporating our system into a cell-free sensor would be extremely difficult because DNA replication machinery would work differently in vitro and overexpressing proteins involved in the SOS response might not be possible. He mentioned that in his experience, it is very difficult to make cells overproduce proteins that are tightly regulated because in high concentrations, these proteins could be toxic to the cells. He also explained that DNA replication is not something that has been done in a cell-free system before and, thus, it would be difficult to get this process to work.
One possibility Adam mentioned was using Recombinase Polymerase Amplification (RPA) to detect DNA damage. This would serve as a yes/no signal, where if a signal is seen, then the DNA was transcribed and was not damaged and if the signal is not seen, the DNA was not transcribed and there was DNA damage. If we were to do this, we would have to make sure that there were sites for thymine dimerization to occur in the GFP gene. The main limitation of this system is that it will not be able to quantify DNA damage and will only provide a yes/no readout answer.
Takeaway: From this meeting, we decided that cell-free was not entirely compatible with our project purpose and decided to design our product to be a whole-cell sensor.
MARKET RESEARCH: WARD SCIENCE KIT
In performing market research to identify any existing educational kits similar to ours, we purchased and used a product from Ward Science, which utilizes ultraviolet-sensitive yeast. We tested this kit when the sun was at its zenith (its highest point) to maximize the damage done to the yeast cells in order to obtain clear results.
We were able to compare the growth of yeast colonies under the conditions of no UV exposure, partial UV exposure, and full exposure. Partial exposure was accomplished by covering one of the plates with aluminum foil, which should've protected the yeast underneath from most if not all of the sun's ultraviolet radiation. After our samples from the kit were placed under direct sunlight for 15 minutes and subsequently removed following the Ward protocol, we let the samples incubate for two days before checking in for final results. In our observation, we had seen that the yeast had grown in the petri dish which was covered by foil, whereas we did not see as dense a culture growing in the yeast plate which received a greater amount of ultraviolet radiation from the sun.
Testing the Ward Science kit demonstrated insight into the user experience when using a product based on learning and scientific exploration. Seeing what came with the kit, such as test tubes, pipettes, and aliquots of water, was incredibly helpful for considering what else would go in our kit. Design analysis of the Ward kit gave us a better idea of how to make our kit engaging and user-friendly. We learned that providing straightforward and simple documentation (a user manual, student worksheet) to go with the kit is essential. By analyzing the Ward kit experimental results we believe that we will be able to improve upon the kit by using our E.coli system which makes use of the NER mechanism to express superfolded-GFP.
Figure 1: Yeast exposed to UV (left) shows significantly less growth compared to yeast that was covered (right).
Check out the Ward Science UV exposure kit here.
OUTREACH: NILES WEST HIGHSCHOOL WORKSHOP
We visited a local high school, Niles West, to introduce students interested in STEM to synthetic biology, our project, and the idea of them having an iGEM team at their school. This workshop included us speaking about synthetic biology and our summer project, showing them different types of fluorescent proteins, and answering their questions on the research process and college.
Many of the students we spoke to at Niles West were very interested in STEM before the outreach event, but many of them had not heard much about synthetic biology previously. Although the topic of genetic engineering can be complex, by introducing real life examples and diving into the endless possibilities of the field, we were able to have a vivid discussion and help students develop a general, but factual, idea of what genetic engineering means. By doing this, we were able to spark an interest and create excitement about the limitless possibilities of synthetic biology.
Here are some pictures from the event.
