Team:Northwestern/Expert Interviews
EXPERT INTERVIEWS
Deciding Between Whole-Cell and 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.