Team:Stanford/Poster



SEED: Self-replicated Environmentally Embedded Diagnostics

Authors: Toby Frager1, Weston Gray1, Emilie Kono1, Chris Neimeth1, Gillian Peraza1, Lauren Ramlan1, Jordan Strasser1, Sarah Yribarren1, Drew Endy2, Stanley Qi2, Jennifer Cochran§.

1iGEM Team Members; 2iGEM Team Mentors; §Faculty Sponsor, Department of Bioengineering, Stanford University.

Abstract

Our team is attempting to engineer a nucleic acid diagnostic in live cells, specifically B. subtilis. B. subtilis is a GRAS, gram positive bacteria that is naturally competent, uptaking ssDNA from its environment. By engineering strains of super-competent B. subtilis with two potential detection systems, our team is testing the detection capabilities of our live cell diagnostic. The first system uses RNA toeholds, riboregulators that target specific nucleic acid sequences. In the presence of the target sequence, the RNA toehold binds, exposing an RBS for ribosome binding and allowing the translation of a reporter protein. The second system repurposes the homologous recombination system found in B. subtilis. In the B. subtilis genome, we engineered regions of homology to a target sequence on both sides of the negative selection marker ManP. When the target sequence is taken into the cell, it recombines into the genome at the homologous regions, excising the negative selection marker and allowing cell growth. We are in the process of testing both of these systems in B. subtilis, and have made the parts we designed available in the iGEM parts registry.

The Motivation: A Self-Replicating COVID-19 Test Kit

When our team started to brainstorm what we wanted to spend this summer creating, we were in the midst of the COVID-19 shutdowns. We had all been watching the pandemic for weeks from our homes, observing shortcomings in our healthcare system that prevented efficient handling of the virus. One of those failures was lack of testing availability, which made isolation and contact-tracing difficult. There were two main factors that led to the problems with testing. The first is that the standard tests require professionals to analyze samples and interpret results, but there are limited personnel available to do so. The second is that testing requires expensive reagents, hardware, and lab equipment - all of which are limited. We wanted to solve these problems by creating a live-cell test that can replicate itself, be grown anywhere around the world, and wouldn't require any expertise or lab equipment to administer or read. By doing this, we could solve both of these problems.


Our Objective: Build a cell that works as a living viral diagnostic, to eliminate testing the widespread shortages illustrated by the graph above.
System Overview: Three Phases From Input To Output

At a high level, there are three phases in the way SEED works:

1. Nucleic acid uptake through natural competence of B. subtilis.
2. Intracellular detection through either RNA toeholds or homologous recombination.
3. Intercellular signaling through the quorum-sensing pathway.

The result is a rapid, colorimetric readout in response to a target.



In creating this system, we developed 8 new parts which can be grouped into three groups: reporters, recombination-based detectors, and toeholds. In addition, we constructed a part for a Signal Amplifier device that was not able to be tested in the lab. These parts were combined to create the two detection systems that we worked in parallel to design and test: detection through in-vivo toehold production and recombination-based detection.

1. Uptaking Environmental DNA

Competent B. subtilis cells express specialized proteins that assemble into a DNA transport complex embedded into the cell membrane. Once the DNA is fully transported into the cell, subtilis will integrate it into its main genome. This is another ability that is unique to Bacillus subtilis. In other competent cells such as E. coli, the eDNA will exist in the cell as a plasmid, separate of the main genome. (Maier et al).


The regulation of competence in B. subtilis is extremely complex. However, two important molecules are ComK and ComX. Expression of the DNA binding, uptake, and recombination proteins necessary for competence are controlled by the transcription factor ComK, which is expressed only when the exponential growth phase ends (van Sinderen et al). The expression of ComK can be stimulated by the pheromone ComX, which is sent from other cells via the quorum-sensing pathway, in response to increasing cell density. (Hamoen et al).

Through understanding the genetic circuits involved with regulation of competence expression, researchers have been able to develop strains with inducible supercompetence in response to a variety of different inputs. In our project, we took advantage of this and used strains that had supercompetence, and were inducible by specific sugars.

2. Detecting The Target Sequence
We tried two different detection methods inside of our cells, one using RNA toehold switches, and the other using B. subtilis' ability to integrate environmental DNA into the main genome through homologous recombination.

RNA Toeholds

Toeholds are strands of RNA engineered in such a way that the strands fold in on themselves to form a hairpin loop. However, when the target sequence comes near to the RNA strand, it binds complementary to the trigger, causing the hairpin loop to unravel. With the RBS now exposed, ribosomes are able to bind to the RNA toehold, resulting in transcription of the reporter.



We designed our toeholds for integration into a plasmid expressing YFP under the pVeg promoter, the strongest constitutive promoter known in B. subtilis, with kanamycin resistance as a selection marker. We plan to insert the toehold sequence into the plasmid right after the pVeg promoter and right before the YFP sequence. Because pVeg is the most active promoter in B. subtilis, this means toehold expression will be maximized.

The trigger strand was designed to be complementary to a section of the KanR gene, which we chose as our target sequence since it was already present on the backbone. For the RBS, we used a sequence specific for ribosome-binding in B. subtilis. The start codon is an optimal distance of 6bp away from the end of the RBS for B. subtilis. For our reporter gene, we chose YFP due to its fast diffusion time and bright yellow fluorescence.

Homologous Recombination

Another approach we investigated is based on the recombination system used in the miniBacillus project and outlined in Altenbuchner 2015. First, we transform a plasmid containing a negative selection marker that is flanked by regions of homology to the target sequence. When the cell takes up the target sequence, a recombination event is triggered, causing the gene encoding the negative selection marker to be excised and replaced by the incoming target sequence.

If cells don’t uptake the target sequence, then recombination does not occur. And if recombination does not occur, then the negative selection maker will remain in the cell, leading to its death when exposed to mannose. Therefore we can tell if the target sequence is present by whether or not the cell dies when exposed to mannose.



The constructs were designed to create a plasmid for integration into B. subtilis at the AmyE gene locus. Using pBS1C as a backbone, our engineered inserts create a ManP selection cassette flanked by homology to the target pOpen_Yeast gene segment. Each region of homology flanking the negative selection maker is around 500 bp. 500bp is the length of homology conventionally used when looking for high efficiency recombination in B. Subtilis, even though some report relatively high efficiency with shorter regions of homology.

For our target sequence, we used a region in the pOpen Yeast plasmid from Free Genes between the AarI and MspA1I cut sites. We used this region because it was readily available to us in quantity, though any sequence of sufficient length could be used as the target.
3. Amplifying The Signal

We built an amplifier by hijacking our cell's native quorum-sensing pathway. Normally, once B. subtilis cells reach a critical population density, their quorum sensing molecules activate global pathways to produce community-wide goods. One such quorum molecule is ComX which, at a critical density, activates pathways that result in the activation of the srfA-promoter (PsrfA). PsrfA activation upregulates the downstream production of surfactant molecules, resulting in a biofilm that is contributed to by every cell in the colony.

The novel pathway we engineered takes the positive feedback loop of ComX from the quorum-sensing pathway, and rewires it to cause cells to produce fluorescent proteins instead of surfactant molecules. The first step was knocking out endogenous ComX in our B. subtilis strain. By doing that, we were then able to put ComX under the expression of whatever promoter we wanted. In the Signal Amplification construct that we designed, the ComX and luxABCDE genes are under the control of the srfA-promoter, PsrfA.

The reasoning is this: once a critical amount of cells are exposed to the ComX molecule, their PsrfA genes are activated to produce even more ComX and luxABCDE (a bioluminescent reporter of our choice). The positive feedback loop would cause bioluminescence to increase exponentially, resulting in a rapid readout.

Modeling

To test and get feedback on our system outside of the lab, we modeled three different aspects of the invention mathematically, in an effort to answer the following research questions:
  • How many cells will take in our target DNA?
  • How do we interpret a measured survival rate for the ManP method of our system?
  • What fraction of positive cases will we detect?
  • What is our false positive rate?
  • How fast do we expect to get a signal using the toehold switch method?
  • How strong do we expect that signal to be?
  • How do we expect results to change when using the ComX signal amplification pathway?


There are two major challenges in predicting the performance of our system. First, the components we are using have not been combined before, so we have a novel set of limiting factors and no existing models to help us understand them. Second, the quantitative information for how each component performs is hard to find and suspect. Here are some answers to the research questions:

  • The number of cells taking in DNA depends heavily on figures for competence in which we aren’t certain. For ballpark estimates, we get a usable ~20% of cells
  • We can graph the probability of target presence. There is a clear cutoff survival rate.
  • In almost all realistic cases, we will detect virtually all positive cases with very high confidence.
  • In almost all realistic cases, we will have virtually no false positives, with very high confidence.
  • The dynamics of toehold switches are hard to model, but simulation in collaboration with Edinburgh iGEM showed that the toehold we designed worked.
  • The total readout level is dependent on the concentration of target DNA, but once we have an estimate for that, we can easily plug it into our model to estimate the signal level of our system.
  • The ComX system is similarly difficult, but once we have figures ComX behaviour and estimates for the toehold system, we can simulate the amplification scheme easily using the tools we developed.

Integrated Human Practices

In our efforts to design the project in ethical and useful way, we conducted 8 separate interviews with professionals involved with many different aspects of COVID testing. Our findings are summarized in these 4 points:

  • Affordability is incredibly important. While we aim to make our technology open-source to make it more accessible to underprivileged communities around the world, insight from our interviews pushed us to focus our short-term plans for implementation on our immediate community where we are well equipped to build positive community relationships.
  • Accessibility means ease of use. This prompted the team to plan a series of experiments to begin validating SEED as an at-home test that is simplistic and adapted to the resources the people have in their homes.
  • Accuracy matters. We were led to design experiments to test the accuracy of SEED after multiple generations of bacteria colonies to validate whether random mutations affect accuracy.
  • Clear and transparent communication of how the technology works, and how well, is vital. We plan to communicate the technicalities of SEED and our experimentation surrounding it from both a scientific and public health lens so that anyone can understand the relevance of our test to them and build their confidence in using it.

    These findings had a very significant impact on our project. For one, the interviews informed experiments to validate simple and easy-to-follow protocols for SEED’s use in at-home settings. But perhaps even more importantly, as our team works to develop SEED beyond iGEM and into the real world, our findings and ethical discussion have a tangible impact on our mission as a group, our goals for the distribution of our test, and other pillars of our entrepreneurial intentions and objectives.

Methodology: Testing In Lab

Our lab situation was a complicated one from the start. Due to the pandemic, we were not permitted to use Stanford's labs. As an alternative, we ended up finding lab space in a biohackerspace / community lab in Santa Clara called Biocurious, where four of our team members were able to work for some length of time. We only got into the lab beginning in September, so we have not been able to fully test our systems. However, here are the methods we are trying.

Toehold Detection System

To test our toehold detection system, we first partnered with the Edinburgh iGEM team to verify the function of our toehold in vitro. We have recently gotten verification that the plasmid works, and are now beginning assembling and sequence verifying the toehold plasmid. We are doing the assembly through first linearizing the plasmid backbone with PCR, and then doing Gibson Assembly with the toehold construct.



Once we have verified that the plasmid has been assembled correctly through sequencing, we will transform B. subtilis with the plasmid. Since the target sequence for our toeholds is contained within the same plasmid, bacteria transformed with the plasmid should express YFP due to toehold binding, giving us the on-level of reporter protein expression.

We will then assemble a new plasmid that does not contain the KanR gene, to test off-levels of expression. Finally, we will assemble a plasmid that has KanR under inducible expression, to measure the change in fluorescence based on when the target is being expressed.

Beyond this, we plan to test toehold binding coupled with uptake of an exogenous target sequence. We also plan to test new target sequences and reporter proteins and expand what the system is capable of.

Recombination-Based Detection System

We are pretty confident, based on the literature, that the homologous recombination system will work. We have no verification to do before we start assembling the plasmid, and so that is our first step. We are doing this by linearizing with restriction digestion, and then assembling with Gibson Assembly.



After sending it in for sequencing to verify, we will transform a plasmid containing ManP that is flanked by regions of homology to the target sequence. Then, we will expose the transformed cells to media with mannose and, if the cells live, then we know that recombination did occur and our system works. If the cells die, we will need to evaluate what went wrong with the system.

Next steps after this is to test the sensitivity and accuracy of detection by adding different amounts of DNA, and sequences that are close to the target but not exact. This will give us more insight into the system's feasibility.

Future Directions

SEED has the potential to be a versatile technology platform with a number of different applications, but because the idea was inspired by COVID-19 testing, we focused on designing an implementation strategy for SEED as a viral diagnostic. In terms of sample collection, the goal is for SEED to function as a non-invasive test, allowing the user to simply expose a saliva or cough sample to the cells in order to get a read-out. This implementation page will focus on that application, moving forward.

1. Validation and Optimization in Lab

Before our invention can be commercialized as a product for public consumer usage, we must answer several questions in a lab based setting. Currently, we are in the Proof-of-Concept stage of research, establishing answers to base questions that will inform what is and isn’t possible for our diagnostic.

2. Clinical Testing and FDA Approval

Before our invention can be commercialized as a product for public consumer usage, we must do clinical trials and testing, as well as get FDA approval. This is a crucial step that is the bridge between the initial proof-of-concept and the actual implementation to bring the test to market.

This step would include testing with patient samples, and setting up the test in a non-laboratory setting. We would further test mutation rates, viability outside of the lab, sensitivity and specificity, and scalability in a clinical setting. Once clinical trials are complete, we would move into FDA and other approval stages.

3. Production and Distribution

B. subtilis has a doubling time of two hours under optimal conditions. Additionally, it can be sporulated and shipped around the world in the mail. Distribution and scaling are easy for this product. We would like to implement a system that allows consumers to specify their desired target sequence and readout, and we would ship them a kit containing their unique cells and all the required reagents to culture and use them - in a kit called "Bac Pac".

Outreach & Education

This year the Stanford iGEM team made a purposeful effort to reach out and start dialogues surrounding synthetic biology with a greater diversity of people. We did this in a variety of ways including a weekly synthetic biology speaker series, regular online lectures about various wet and dry and lab techniques, and a high school mentorship program that enabled 21 high school students to work in research teams to design free-use versions of a protein of interest.

SynBio Speaker Series: Innovative, Inspiring Talks From Experts Across The Field

Our SynBio Speaker Series consisted of a series of hour-long talks hosted weekly every Thursday at 4 PM PST on Zoom, open to anybody who had the link and wanted to join. They are now currently on our YouTube channel for anyone to learn from and have received hundreds of views collectively since being posted online.

Lab Webinars: A Virtual Way To Teach Lab Techniques

The webinars function as an introduction to wet and dry lab techniques that are easily accessible for beginners to understand. They were hosted weekly every Tuesday at 4 PM PST on Zoom, and are on our YouTube channel along with the SynBio Speaker Series videos.

Mentorship Program: An Online Program To Enable High Schoolers To Implement Independent Research Projects
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With our High School Mentorship Program, we wanted to bring high school students their own miniature iGEM experience; an opportunity to work in teams and execute an independent research project, with the aim of solving a relevant problem in the world. As they worked through their projects - which was in the realm of protein engineering - we were able to teach them more about biology, patent law, and bioinformatics, and to take them through how to implement a research project from start to finish.



Many proteins that are incredibly useful in scientific research and commercial application are patent-protected, and require a licensing fee be paid to the patent-holder whenever they are used. This can be a financial burden on people trying to use the protein. The project we mobilized our high school students to solve involved the following steps:

  1. Identifying a patent-protected protein that would be useful if it was free-use
  2. Breaking down the patents surrounding the protein
  3. Compiling and aligning sequences of related proteins
  4. Generating mutant sequences based on the aligned sequences
Results

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.



Conclusion: From the experiments we ran, we know that a detection system in B. subtilis could be a reliable way to assay for nucleic acid sequences as we were able to confirm that B. subtilis has the ability to reliably take in and integrate sequences into its genome. We were also able to determine that competence and transformation could be done easily with minimal equipment following a simple procedure, which shows it can be used as a potential at-home test. The most exciting results that we received was showing that the toehold construct we designed works in vitro. This means that once we transform into our cells, the toehold should also work in vivo unless it is not possible for toeholds to work in subtilis. Showing that our toehold can work in B. subtilis would be novel and is the goal we are working towards based on the results we have.
References and Acknowledgements

A huge thank you to everyone who supported us this summer as we worked on our project. You were all incredibly helpful for us on our journey as we navigated the research process, and we really appreciate everything you did to help us get the most out of our iGEM experience.

Acknowledgements

Drew Endy
Stanley Qi
Daniel Zeigler
Elizabeth Libby & Pamela Silver
Josh Tycko
Peter Cavanagh
Keoni Gandall
Alec Lourenco
Eesha Sharma
Biocurious
Stanford Medicine Catalyst
Gary Olsem

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