While implementing the design-build-test cycle in our lab this year, our team implemented all safety precautions and considerations that were available to us. 1) Our university, The College of William and Mary, has a rigorous Safety Program and we worked closely with the Safety Office throughout the entire iGEM season. 2) Our team underwent a wide range of training, provided by both our university and iGEM. 3) All experiments conducted throughout our project only pose minimal risks associated with routine BSL1 laboratory work. The genetic circuits we constructed do not produce any compounds that we anticipate could cause health risks. All hazardous chemicals were handled appropriately according to our university’s laboratory and research safety guidelines and our Institutional Biosafety Committee (IBC) protocol, and all COVID-19 preventative guidelines were followed. 4) Upon analyzing the dual use of our devices, we have concluded that the risks are very low, both in likelihood and impact. 5) Most importantly, our project as a whole addresses an essential safety feature: orthogonality. Our project aims to assess orthogonality between a circuit and its host, and therefore directly addresses major safety concerns due to circuit unpredictability.
1. Institutional Safety Measures
Although our iGEM laboratory is designated for BSL 2 work (and we have been approved for BSL 2 work by our IBC protocol), our iGEM project this year operates totally at BSL1. By definition, according to NIH/CDC regulations, we are not working with any organisms which typically cause diseases in healthy humans. However, we nevertheless take all possible precautions. To prevent transmission or escape we use best practices as outlined by the CDC, NIH, and the William & Mary Institutional Biosafety Committee.
We take the following precautions:
- The laboratory is not accessible to anyone but those working on the project who have received training. It is also locked with a private passcode.
- Recombinant DNA and bacterial cultures are stored in -80°C and -20°C freezers in the Bioengineering Laboratory (Integrated Science Center Room 1227), which has restricted access to those who have been specifically trained and approved.
- Members must wear long pants and/or lab coats for protection.Close-toed shoes are required at all times in the lab, and gloves whenever working at the bench. Lab coats are required when working with BSLreagents. Goggles are required when working with any caustic or BSL reagents that could readily aerosolize. Students utilize heat-safe gloves when working with the autoclave and with glassware on the hotplate. Any protective clothing worn is always left in the laboratory or, if disposable, always placed in trash that will be autoclaved in secure bags using a certified autoclave.
- All trash materials are thoroughly disinfected via 10% bleach and/or a certified autoclave before leaving the premises. All bacterial cultures are sterilized with 10% bleach. All BSL1-contaminated materials are placed into a bin labeled “BSL1 trash” lined with an autoclave safe bag before autoclaving. Bags are then sealed with autoclave tape and autoclaved on the Gravity 20 Cycle (20 minutes at 121°C). Following successful sterilization, BSL-1 trash bags are immediately disposed of in the trash container in the loading dock of the Integrated Science Center (ISC).
- Flasks and other glassware used for bacterial cultures are soaked in 10% bleach before final disposal down the sink, followed by flushing with tap water. All glassware is washed thoroughly with hot water and detergent, rinsed in DI water, and baked for sterility. Disposable glassware is thrown away in designated cardboard containers for broken glass.
- All bench space is wiped down with 70% ethanol before and after use with BSL1 material.
- No food, drink, or cosmetics are allowed in the laboratory. Hands must be washed before leaving the laboratory.
There are many levels of safety and oversight at our institution and our iGEM team has engaged with each one. Teresa Belbeck, our Safety Officer, is responsible for overall lab safety at William and Mary. Working with her is Crystal Taylor, whose office is in the Integrated Science Center (ISC) and who works very closely on a daily basis with the labs in our building. She also conducts regular inspections. Both of our Safety Officers are highly trained with extensive experience in laboratory safety. They are very familiar with all aspects of the procedures and practices of our project. In addition, we submit our protocol to the William and Mary Institutional Biosafety Committee, a group of faculty experts and experts in lab safety from the community. Our protocols are reviewed and approved, and we are provided with any suggestions or comments.
Dr. Margaret Saha (Chancellor Professor of Biology) oversees all aspects of the project and has around 30 years of experience working with recombinant DNA and BSL1 and BSL2 infectious agents. She has served as PI of William and Mary labs with undergraduates and graduate students since 1993 and of W&M iGEM teams since 2014. All team members who worked in a lab setting received training from Dr. Saha in basic chemical safety and biosafety (BSL1).
In addition, all team members received two hours of training on all aspects of lab safety from the Director of the Environmental Health and Safety Office at the College of William and Mary. Materials that were covered include mechanical, chemical, radiation, and biosafety, as well as basic laboratory safety information such as attire, organization, hazard symbols, and prevention/handling of laboratory incidents. All safety training sessions were documented.
New lab members received training from Dr. Margaret Saha or other senior personnel whenever learning a new protocol. This applied to simple lab procedures like pipetting, waste removal, working under the fume hood, as well as protocols for procedures such as minipreps and restriction digests. Additional training was carried out by Dr. Margaret Saha and other senior personnel on a continuing basis.
Protocols contain detailed instructions and alert members to possible safety risks. All protocols are consistently updated and posted on the William and Mary iGEM 2021 team Google Drive. Members attended specific training sessions when using certain equipment, such as autoclaves and the ultracentrifuge.
As required for the collection of data on human subjects, which is necessary for human practices, our team completed virtual training through the online CITI training program for the following modules: “Biomedical Research Investigators - PHSC” and “AREA III Disciplines - Research Ethics.” Members of our team have also attended the “Safety Workshop: Dual-Use Research of Concern” held by iGEM this year and participated in the Dual Research of Concern Risk Assessment Study, further familiarizing ourselves with the concept.
3. Safety in Design and Circuit Testing
Safety in Design
In order to mitigate any risks associated with the orthogonality portion of our project, our team is following all BSL1 safety guidelines and is only using nonpathogenic strains. Additionally, by not releasing our engineered bacteria into the environment, we are able to avoid the risk of gene transfer with native organisms.
We worked with Escherichia coli BL21 (B line derivative), Escherichia coli BL21(DE3) (B line derivative that contains the λDE3 lysogen, which carries the gene for T7 RNA polymerase under control of the lacUV5 promoter), Escherichia coli DH5-alpha, Escherichia coli NEB 5-alpha cells (K-12 DH5-alpha derivative with T1 phage resistance and endA deficiency), and Escherichia coli JS006 (a MG1655 derivative that has lacI and araC knocked out; K-12 derivative) all of which are BSL1. E. coli K-12 and B laboratory strains are well-characterized and not known to be pathogenic in immunocompetent individuals (NIH, 2019). We followed the appropriate safety practices for handling these organisms in accordance with the recommended practices of the NIH as well as our institution. A full list of safety practices is provided within our safety form.
However, we would like to note that our experiment involves transforming several BSL1 E. coli strains with plasmids containing resistance to the following antibiotics: kanamycin, ampicillin, spectinomycin, chloramphenicol, and streptomycin. If these organisms managed to escape into the environment, they could pass antibiotic resistance genes to their wild type counterparts. However, because of the aforementioned safety measures we implement, no infectious organism leaves the laboratory except after being autoclaved or disinfected with 10% bleach.
The primary vector we will be using is one of the pSB vectors developed by iGEM. These vectors contain restriction enzyme cut sites (for EcoRI, XbaI, SpeI, and PstI) in Prefix and Suffix regions that flank the functional insert of interest (Shetty et al., 2008). The plasmids are characterized on four different antibiotic cassettes for ampicillin, chloramphenicol, kanamycin and tetracycline. For all circuits we have constructed, we used the pSB1C3 vector flanked by unique nucleotide sequences (UNS) 1 and 10 (Halleran et al., 2018), which is a high copy plasmid with a mean copy number of approximately 100-300, and a standard plasmid backbone on which circuits developed by iGEM teams are typically constructed.
The majority of parts that we have chosen to use this year are parts that have been previously characterized by previous iGEM teams and have been shown to not be harmful to humans, animals, or plants. Of the novel sequences we submitted, none encode any toxins or proteins that have been shown to be harmful to higher organisms. It is not anticipated that the translated products of any of the circuits we have tested will cause any health risks to humans, animals, or plants.
The transcriptional sensor circuit used in our project, pUC19-P70a-3WJdB-T - Addgene #87311, designed by researchers Alam et al., places transcription of the three-way Junction dimeric Broccoli (3WJdB) aptamer under the control of the constitutive p70a promoter, and is an RNA aptamer that fluoresces when bound to the fluorophore DFHBI-1T. Our translational sensor circuits (WM21_013 and WM21_016) encode a gene for sfGFP, which is constitutively expressed. Our phosphorylation sensor circuits (WM21_014 and WM21_015) also encode the sfGFP gene, expressed upon induction by high osmolarity. Our glycosylation sensor circuit (WM21_022) encodes the sfGFP gene, expressed upon induction by the bacterial SOS response. Our heat shock sensor circuits (WM21_017, WM21_018, and WM21_021) encode the sfGFP gene, expressed upon induction by the heat shock response. Our AG43 sensor circuits (WM21_019 and WM21_020) encode the sfGFP gene, expressed constitutively. Our protease sensor circuits (WM21_023 and WM21_024) encode the sfGFP gene, expressed upon induction by the heat shock response. All circuits constructed by our team exclusively express fluorescent markers, either in the form of a green fluorescent protein or an RNA aptamer that fluoresces upon binding to fluorophore DFHBI-IT, neither of which has any known harmful effects to humans, animals, or plants.
Safety in Circuit Testing
Throughout the circuit testing process, our team followed the guidelines in our university-approved Institutional Biosafety protocol for all lab work conducted for this project. In addition, we did not work with circuits containing any genes encoding for proteins that could harm humans, animals, plants, and have intended use of our toolkit to be restricted to the laboratory environment. Therefore, the only risks associated with its use are the minimal risks corresponding to routine BSL1 lab work.
For collection of RNA samples, our team worked with the following plasmids: pDAWN-Ag43 - Addgene #107741 (Jin et al., 2018), pBbB8k-csg-amylase - Addgene #166859 (Birnbaum et al., 2021), pCDF_LuxR - Addgene #140039 (Barbier et al., 2020) and TS_pLuxLac - Addgene #140426 (Barbier et al., 2020). After transforming pDAWN-Ag43 into E. coli JS006, the functionality of this circuit was confirmed using biofilm lithography followed by a crystal violet staining procedure, as described by researchers Jin et al. (Jin et al., 2018). After transforming pBbB8k-csg-amylase into E. coli DH5-alpha, the functionality of this circuit was confirmed using a Congo Red spin down assay developed by the 2017 Harvard iGEM team (Harvard iGEM Team 2017). After co-transforming E. coli BL21 with pCDF_LuxR and TS_pLuxLac, the functionality of this system was confirmed by measuring fluctuations in green and red fluorescence of host cells in the presence of either AHL or IPTG using a plate reader.
For the sensor circuits designed by our team to quantify transcriptional and translational burden along with post-translational modifications in vivo, as well as the production of orthogonality markers such as heat shock proteins, proteases, and AG43 in vivo, we used Gibson Assembly for construction of our circuits. We designed each of our circuits to contain unique nucleotide sequences (UNS) 1 and 10 (Halleran et al., 2018) before the promoter and following the terminator, allowing for the assembly of our circuits into the backbone pSB1C3 flanked by the same UNS sequences, 1 and 10 (pSB1C3 1/10). Our circuits were synthesized by IDT as a single gBlock containing the UNS sequences required for assembly with pSB1C3 UNS 1/10 through Gibson Assembly. After transforming our circuits into several BSL1 strains of E. coli, we conducted colony PCR to confirm that our circuits were successfully constructed. We confirmed the functionality of our circuits by measuring fluorescence using a plate reader.
Most of our circuits contain a codon optimized version of the sfGFP gene, which is a synthetic version of the gfp gene from Aequorea victoria. For our circuits designed to test protease production, we used the clpB promoter from E. coli K-12 derivative strain H12017, the clpB RBS from E. coli K-12 derivative strain H12017, the lon promoter from E. coli K-12 strain RGC121, and the hslVU promoter from E. coli K-12 MG1655. For our circuit designed to quantify glycosylation, we used the ftsL promoter from E. coli K-12 strain JM103. For our circuits designed to test biofilm formation, we used the flup1 and flup2 promoters from E. coli K-12 MG1655. For our circuits designed to test heat shock protein production, we used the dnaK promoter from E. coli K-12 SC122. All circuits express sfGFP except for those designed to sense transcriptional burden. For the in vivo transcriptional burden sensor, we are utilizing the three-way Junction dimeric Broccoli (3WJdB) - Addgene #87311 (Alam et al., 2017) aptamer under the control of the p70a promoter, which binds to the membrane-permeable, non-cytotoxic dye DFHBI-1T. After incubation of our chasses (transformed with the transcriptional burden sensor) with this dye after overnight growth, we will be able to quantify fluorescence and OD600 using a plate reader. For one of the in vivo translational burden sensors we are testing, designed by Ceroni et al. (2015 - Nature Methods), we are using promoter BBa_J23100 along with a synthetic RBS designed by Ceroni et al. using an RBS calculator to control expression of sfGFP. For the other in vivo translational burden sensor, we are using promoter BBa_J23119 along with RBS B0034 to control expression of sfGFP.
As alternative approaches to our circuits designed for in vivo use, we designed and constructed circuits for use in a cell-free system as well as integrated our circuits designed for in vivo use into the genomes of our chasses (E. coli NEB 5-alpha, E. coli BL21(DE3), and E. coli DH5-alpha) using the lambda red system. Specifically, we are conducting genome integration through the use of plasmids pCas - Addgene #62225 (Jiang et al., 2015) and pTargetF - Addgene #62226, (Jiang et al., 2015). For our circuits designed to measure transcriptional and translational burden in vitro, we used the p70a promoter from bacteriophage lambda to control the production of sfGFP and the Spinach2 aptamer. After construction through Gibson Assembly, our circuits were co-transformed with plasmid pBbB8k-csg-amylase to assess various aspects of its orthogonality. We also tested plasmids designed by Ceroni et al. (Ceroni et al., 2018) to test heat shock protein production in E. coli after transformation with our sensor circuits and after cotransformation with our sensor circuits and pBbB8k-csg-amylase. The names of these plasmids are groSL-GFP - Addgene #109389, htpG2-GFP - Addgene #109388, htpG2-GFP - Addgene #109388, and ibpAB-GFP - Addgene #109390 (Ceroni et al., 2018). Plate reader measurements of fluorescence and OD600 were taken at the zero, one, six, twelve, twenty-four, and forty-eight hour time intervals. Additionally, our team performed qRT-PCR to confirm the functionality of our circuits. Finally, we analyzed the autofluorescence levels of our chasses before and after transformation with our sensor circuits, and before and after cotransformation with our sensor circuits and pBbB8k-csg-amylase using a fluorescence microscope as an alternative method for assessing orthogonality.
In order to confirm the efficacy of this system, our team is homogenizing cells transformed with pBbB8k-csg-amylase using freeze/thaw cycles and mechanical disruption, and adding the cell lysate to centrifuge tubes containing our sensor circuits. Plate reader measurements of fluorescence and OD600 are then taken at the zero, one, six, twelve, twenty-four, and forty-eight hour time intervals. In conclusion, our circuits do not contain any genes encoding proteins that could cause harm to humans, animals, or plants, and are not intended for use outside of the laboratory environment, and experiments done using our circuits pose no risks other than the minimal risks associated with routine BSL1 lab work.
This year, our project required the use of ethidium bromide (a mutagen) and hydrochloric acid and sodium hydroxide (a corrosive chemical). Team members were instructed to use the fume hood when creating agarose gels with ethidium bromide and to dispose of the gels in a hazardous waste container that is collected by the Environmental Health and Safety Office at the College of William and Mary. Additionally, protective clothing was worn when handling hydrochloric acid and sodium hydroxide.
In alignment with the recommendations given by the CDC, The College of William and Mary has required all students and faculty to wear masks indoors to mitigate the transmission of the SARS-CoV-2 virus. This requirement was in effect from the start of the year until June 22, and resumed on August 10. Our team has worn masks during any iGEM-related activity that involved being in the presence of other people indoors (examples include: meetings, working in the laboratory, outreach activities, and in-person collaboration events). We also practiced healthy habits, such as hand washing, surface sanitization, and physical distancing.
4. Dual Use
Given that our toolkit is intended for use only in a laboratory setting, there is minimal risk associated with its use. However, due to the incorporation of antibiotic resistance genes within all of the sensor circuits in our toolkit, there is a risk of horizontal gene transfer with other organisms. Antibiotic resistance genes could potentially bolster the threat level of pathogenic bacteria by making them more difficult to eliminate if our engineered bacteria were to escape the laboratory environment. There is also the potential that our sensor circuits could spread autonomously in the environment due to the incorporation of antibiotic resistance genes within our plasmid vectors. However, as our circuit testing system is not designed to be used in environments outside of the laboratory, our team assumes that other teams and synthetic biologists will follow all required BSL1 safety procedures to prevent bacteria engineered with our circuit system from escaping the laboratory environment. If these procedures are followed, there is little chance of these engineered bacteria spreading autonomously in the external environment.
We intend to integrate our sensor circuits into the genome of E. coli DH5-alpha, creating a test strain to assess several aspects of the orthogonality of the end user’s circuit on the host cell. We believe that with the biosecurity regulations implemented by William and Mary (as mentioned above), it would be difficult for our strain to escape the laboratory environment. In addition, our method of genome integration does not involve antibiotic resistance, so there are no resistance genes that could be passed onto native organisms in the external environment if our engineered strain were to escape the laboratory environment.
5. Orthogonality as an Essential Safety Feature
Our project as a whole is central to the safety of all synthetic biology circuits. Through the creation of an accessible toolkit designed to assess the orthogonality of genetic circuits to their host cells, we are addressing the safety concerns associated with circuits that lack orthogonality and therefore may not function as intended. Orthogonality is defined as the lack of unintended interactions among parts of a circuit, between a circuit and host. Quantifying these unintended interactions is crucial to ensuring the safety of genetic circuits, as these interactions not only decrease circuit efficacy, but also raise major safety concerns due to circuit unpredictability. To ensure the safe application of genetic circuits in the real world, the orthogonality of all genetic circuits requires thorough investigation. However, this relies upon the development of a highly accessible method of orthogonality assessment. Our project this year attempts to address this need through the creation of a toolkit designed to assess orthogonality at the circuit-host level. This toolkit consists of a circuit system and a multilevel model. Our circuit system is designed to assess certain aspects of orthogonality including transcriptional, translational, and post-translational burden as well as the production of orthogonality markers identified through an intensive analysis of published RNA-sequencing data, through which we analyzed changes in host gene expression before and after transformation of bacterial cells with a circuit. Using our multilevel model, iGEM teams and synthetic biologists can input measurements taken using our circuit system in order to gain an orthogonality assessment for their circuits. Based on the assessment provided by our model, teams will be able to improve the safety and efficacy of their circuits through identifying the aspects of host-circuit orthogonality that may be lacking for their circuits. In addition, our system is widely accessible by iGEM teams and synthetic biologists, as our model is compatible with several kinds of data gained through various levels of measurement, such as RNA-sequencing, qRT-PCR, use of our circuit system, and quantification of host cell autofluorescence. We envision our system as a stepping stone to the formation of a well-established, standardized orthogonality assessment method at the circuit-host level in the synthetic biology community—essential to maximizing circuit efficiency, modularity, fieldability, predictability, and safety.
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