At-home, cell-free synthetic biology education modules for transcriptional regulation and environmental water quality monitoring

As the field of synthetic biology expands, the need to grow and train science, technology, engineering, and math (STEM) practitioners is essential. However, the lack of access to hands-on demonstrations has led to inequalities of opportunity and practice. In addition, there is a gap in providing content that enables students to make their own bioengineered systems. To address these challenges, we develop four shelf-stable cell-free biosensing educational modules that work by just-adding-water and DNA to freeze-dried crude extracts of Escherichia coli. We introduce activities and supporting curricula to teach the structure and function of the lac operon, dose-responsive behavior, considerations for biosensor outputs, and a ‘build-your-own’ activity for monitoring environmental contaminants in water. We piloted these modules with K-12 teachers and 130 high school students in their classrooms – and at home – without professional laboratory equipment or researcher oversight. This work promises to catalyze access to interactive synthetic biology education opportunities.

and 24 hours for each reaction set. Researchers then qualitatively assigned to each photograph 2 3 5 values 0, 1, or 2 to represent "OFF", "FAINT", or "ON", respectively. The indicated bars 2 3 6 represent the average score from 21 student replicates of the reporter activity at each time point 2 3 7 (indicated by bar shading), for each reporter (indicated by bar color), and in white and blue light 2 3 8 (left and right plots, respectively). One sample time-course with paired photos is shown. The students' data generated in the classroom matched the laboratory data very well.

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Dispensing either water or IPTG solution from micropipettes, the students generally achieved 2 4 3 the expected qualitative results in Modules 1 and 3; somewhat greater variability was observed 2 4 4 Module 2, possibly due to errors in serial dilution (the students performed their own dilutions 2 4 5 . CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2023. increase in mRFP production between tubes 1-6 as IPTG dose increased.

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To quantify success rates in Module 3, since we could not take plate reader 2 5 1 measurements at intermediate timepoints in the classroom, we instead assigned qualitative 2 5 2 brightness scores of 0, 1, or 2 to each reporter, with and without the inducer, at each timepoint, 2 5 3 based on the students' photographs in white and blue light. After one hour, most students 2 5 4 observed the sfGFP and Mango reporters to be ON in blue light, and the XylE reporter was ON 2 5 5 in white light. Increases in both signal and leak were observed for all reporters after 24 hours. In 2 5 6 many cases, the XylE signal was stronger in the OFF state than the ON state after 24 hours 2 5 7 because the product of the chemical reaction degrades. To assess whether the biosensing kits were effective in inspiring and teaching the 2 6 1 students who did the activities in the small-scale implementation, we measured how well the 2 6 2 program goals were met through pre-and post-module surveys ( Figure 3A). The survey 2 6 3 consisted of a series of statements and asked the student to rate whether they agree with the 2 6 4 statement or not on a scale from 1-5, with a score of 1 indicating that they strongly disagree with 2 6 5 the statement, a score of 3 indicating that they neither agree nor disagree, and a score of 5 2 6 6 indicating that they strongly agree with the statement. Prior to completing any of the three 2 6 7 experimental activities, students were asked to fill out a survey to establish baseline biology 2 6 8 knowledge and perceptions. Following each of the experimental activities, but before seeing any 2 6 9 material for the next activity, students were asked to take the same survey again to capture 2 7 0 changes as a result of participating in that module. . CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2023. ; https://doi.org/10.1101/2023.01.09.523248 doi: bioRxiv preprint 2 7 3  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2023. ; https://doi.org/10.1101/2023.01.09.523248 doi: bioRxiv preprint 0 solve problems, answer questions, and formulate questions of their own [1, 65, 66], we next 3 0 1 created an avenue for students to design, build, and test their own cell-free biosensors. As a 3 0 2 model, we developed a biosensor activity to detect water contaminants of public health concern.
3 0 3 Previous efforts to engage students in the synthetic biology design-build-test-learn 3 0 4 framework have struggled with the build phase due to challenges in DNA assembly and    Comparing the leak, dose response, and stability of response of the copper sensor with aptamer and enzymatic reporters 6 Measuring the dose-response of fluoride sensor with enzymatic reporter output 7 Detecting copper in environmental water samples 8 Measuring the specificity and kinetics of the lead sensor with the aptamer output at 26 and 37 °C 9 Measuring the limit of detection of the lead sensor with the enzymatic reporter 3 3 9 Student success in Module 4 was varied, and many groups reported explicit sources of 3 4 0 experimental error in their lab reports (e.g., contamination, loss of the lyophilized reaction 3 4 1 pellet). However, this study provides a powerful proof-of-principle for the versatility of cascaded 3 4 2 . CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2023. ; https://doi.org/10.1101/2023.01.09.523248 doi: bioRxiv preprint cell-free sensors, which allow students to detect any target molecule with any reporter output, control of the o-T7 promoter. Finally, they were given liquid stocks for copper, lead, and fluoride.

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By rehydrating the sensor reaction with a reporter plasmid and analyte of choice, students could 3 6 1 therefore build, test, and design arbitrary sensor-output pairs for common inorganic water (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2023. ; https://doi.org/10.1101/2023.01.09.523248 doi: bioRxiv preprint Figures 2 and 3 were performed in high school classrooms, but these were still equipped with 3 7 9 scientific instruments such as micropipettes. When many American high schools shut down in 3 8 0 2020 due to the COVID-19 pandemic and biology classrooms switched overnight to remote 3 8 1 learning, we wondered if the intrinsic safety (i.e., no living cells) and thermal stability of freeze-3 8 2 dried cell-free sensors would allow them to be used for at-home experiential learning.

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To test this possibility, we assembled the largest-scale distributed cell-free expression . CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2023. ; https://doi.org/10.1101/2023.01.09.523248 doi: bioRxiv preprint 3 9 7  The results (Figure 5B) from the distributed, large-scale Module 1 were less consistent 4 1 6 than what we previously observed at the small scale (Figure 2A). Surprisingly low expression of 4 1 7 mRFP was observed from both pairs of conditions that used the pLac-mRFP plasmid. In many (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2023. ; https://doi.org/10.1101/2023.01.09.523248 doi: bioRxiv preprint activation. Among the subset of tubes where mRFP production was visible at endpoint, nearly 4 2 0 all students did obtain constitutive expression from tubes 3 and 4 (J23119-mRFP) and observed 4 2 1 IPTG-mediated induction in tube 6, compared to tube 5.

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Overall, by scoring each tube's brightness level as "OFF", "FAINT", or "ON", we 4 2 3 assessed around a 60% global success rate for the module, which we considered acceptably 4 2 4 comparable to standard biology and chemistry classroom labs. We could not easily ascertain 4 2 5 the origin for the failure mode at scale. However, when the remaining reactions were rehydrated 4 2 6 by experienced biology teachers using micropipettes, the constitutive reactions activated well, 4 2 7 and the fixed-volume pipettes also worked, although they were less accurate ( Figure S5A).

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There were also several examples of student tubes that were clearly over-diluted with water or 4 2 9 IPTG relative to the nominal pre-lyophilized volume. These effects do inhibit protein synthesis 4 3 0 ( Figure 5B, Figure S5B).

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Considering the scale and purpose of the experiment, we did not repeat it. We instead 4 3 2 emphasize that further work should be done to investigate the reproducibility of cell-free  aptamer, or a colorimetric enzyme) for a more creative synthetic biology experience. Finally, we 4 5 2 scaled up Module 1 (mechanism) for field testing with deployment to >100 students at schools 4 5 3 700 miles away and achieved ~60% success across all modules using disposable pipettes in 4 5 4 . CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2023. ; https: //doi.org/10.1101//doi.org/10. /2023 classrooms and home settings during the SARS-CoV-2 pandemic. The effectiveness of these 4 5 5 modules was assessed through surveys before and after each set of experiments, revealing 4 5 6 significant increases in several perspective and comprehension questions. Students reported 4 5 7 increased understanding of biological sensors and reporters and felt that classroom labs were 4 5 8 based on modern science after completing the modules.

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Teaching principles of genetic regulation, biological sensing mechanisms, and field 4 6 0 applications of synthetic biology in a hands-on fashion has the potential for significant impact. In      Star (DE3) was inoculated into 1 L of 2X YT+P media (16 g/L tryptone, 10 g/L yeast extract, 5 5 1 1 g/L sodium chloride, 7 g/L potassium phosphate dibasic, and 3 g/L potassium phosphate 5 1 2 monobasic adjusted to pH 7.2) and grown, shaking at 220 rpm at 37 °C, to optical density 3.0 5 1 3 . CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2023. ; https://doi.org/10.1101/2023.01.09.523248 doi: bioRxiv preprint