Safety

When designing and constructing the parts of our device, we prioritized safety in our protocols and practices. Researchers wore latex gloves when working with E. coli and B. subtilis. All lab spaces and equipment were wiped down with ethanol immediately before and after experimentation to prevent contamination. Any used plates, gloves, pipette tips and wipes were properly disposed of in biological hazard containers. Our lab is equipped with features which protect researchers experimenting with the parts we have designed. This includes multiple specific waste disposal bins, a regularly checked fire extinguisher, and fire sprinklers, an AED, and a first-aid kit. A fire evacuation plan was discussed and is displayed in case of a fire emergency. Because our lab generated chemical and biological waste, we took the necessary precautions to prevent contaminating our facilities or the outside environment. We exercised the appropriate waste disposal guidelines outlined by the United States Environmental Protection Agency (US-EPA) [1]

We chose a non-pathogenic chassis for our device. Bacillus subtilis is a benign organism that is not considered pathogenic or toxigenic to humans, animals, or plants [2]. All of the DNA used within the parts is native to B. subtilis or Homo sapiens. No risks were identified in association with the genes corresponding to these proteins related to mutation, overexpression, or underexpression. Therefore, none of the parts utilized in construction of our device present any risk to humans and potential patients consuming our device. To contain our device to the desired environments, additional features were designed for our device in the form of the dual control kill-switch.

We designed a dual control safety mechanism using a toxin/antitoxin system that prevents the bacteria from surviving if excreted from the body. Since B. subtilis thrives in soil [3], this biocontainment mechanism minimizes the risk of environmental contamination. The bacteria will only live in physiological temperatures or if the sugar molecule xylose is present. Our mechanism was inspired by the 2023 NMU-China team’s MazE/MazF kill-switch, which was dependent on rhamnose and temperature [4].

The following circuit diagrams depict our design for the biocontainment mechanism.

Figure 1. Circuit diagrams depicting the individual parts and construct for the biocontainment mechanism.

Part Justifications:

• Xylose Induced Antitoxin
• Pxyl Promoter: Pxyl is a xylose-inducible promoter for B. subtilis. When xylose is present, it binds to XylR, preventing XylR from repressing Pxyl and allowing for expression of MazE5 [5].
• RBS: This is the consensus RBS sequence for B. subtilis, which is used to facilitate high expression of downstream genes [6].
• MazE: Inhibitory antitoxin for the MazF toxin. Our MazE and MazF are derived from E. coli and are commonly used in antitoxin/toxin systems in B. subtilis and E. coli. When this protein is expressed, the cells stay alive [7].
• B0015 Terminator: This terminator is commonly used in and optimized for B. subtilis. It is the same terminator used in our main genetic circuit containing lipase and the switch mechanism [8].
• Temperature Sensitive Component
• P43 Promoter: P43 is a constitutive promoter for B. subtilisthat allows for efficient and continual downstream gene expression during all phases of bacterial growth [5].
• RBS No-chill 06: RBS No-chill 06 is an RNA thermometer, meaning that it changes conformation with different temperatures to regulate translation. Between 25°C and 37°C, it folds into a hairpin loop that obstructs the binding site, preventing the ribosome from binding and thus preventing translation. Above 37°C, the RNA thermometer “melts” and unfolds to expose the binding site for the ribosome to initiate translation of MazE [9].
• MazE: Inhibitory antitoxin for the MazF toxin. Our MazE and MazF are derived from E. coli and are commonly used in antitoxin/toxin systems in B. subtilis and E. coli. When this protein is expressed, the cells stay alive [7].
• RBS: This is the consensus RBS sequence for B. subtilis, which is used to facilitate high expression of the downstream gene MazF [6].
• MazF: A stable, sequence-specific endoribonuclease toxin that is derived from E. coli. MazF initiates a programmed cell death pathway when MazE is not present. In our pathway, MazF is always produced and is inhibited by MazE when either xylose is present, or the temperature is at or above 37°C [10].
• B0015 Terminator: This terminator is commonly used in and optimized for B. subtilis. It is the same terminator used in our main genetic circuit containing lipase and the switch mechanism [8].
• Xylose Repressor
• Note: This part is designed to go in the plasmid in the opposite direction of parts 5a and 5b. The xyl operon which is native to B. subtilis and all recombinant systems we’ve reviewed in literature using parts of the xyl operon have the parts arranged in this way.
• P43 Promoter: P43 is a constitutive promoter for B. subtilis that allows for efficient and continual downstream gene expression of XylR during all phases of bacterial growth [5].
• RBS: This is the consensus RBS sequence for B. subtilis, which is used to facilitate high expression of the downstream gene XylR [6].
• XylR: This encodes for the Xyl regulatory protein which constitutively represses Pxyl by binding to a XylR operator when xylose is absent. Our XylR operator is embedded in the promoter sequence. When xylose is present, it binds to XylR, preventing it from binding and repressing Pxyl and thus allowing for translation of MazE antitoxin in part 5a [11].
• B0015 Terminator: This terminator is commonly used in and optimized for B. subtilis. It is the same terminator used in our main genetic circuit containing lipase and the switch mechanism [8].

The table below shows the condition of the bacteria in different locations. Our switch ensures that the bacteria are alive while being transported in a xylose-containing yogurt. Xylose is a natural sugar that is safe to consume but is not as common in the environment as glucose. When the yogurt is ingested and the bacteria passes through the stomach, it is in the presence of both xylose and the physiological body temperature of 37°C, so it will also survive. When traveling through the intestines, there will be no more xylose present, but the temperature will still be appropriate to keep the bacteria alive. When excreted, however, both xylose and physiological body temperature are absent, and the bacteria is not viable.

We initially planned to construct our biocontainment switch using traditional assembly methods. However, due to an oversight during parts design, along with obstacles with ligation, we were experimentally unable to perform this assembly successfully.

We designed primers to attach Gibson sequences to our biocontainment-switch using PCR. We successfully performed PCR and were able to attach these Gibson sequences to the ends of our parts. We are currently in the process of preparing for Gibson assembly.

We plan to test our biocontainment mechanism using a viability assay. Our cells will be placed in the four environments as described in Table 1, and their condition will be measured using a cell-staining viability assay.