Overview:

Throughout the timeline of our project, PHAntom 2025 recognized the importance of following proper safety protocols in the lab setting. We made a commitment to following established safety procedures and guidelines to ensure the welfare of our team and environment while conducting research. Our team aimed to minimize risk to prevent accidents before they could happen. By adhering to guidelines set forth by the University of Rochester’s Environmental Health and Safety department standards, we were able to reduce these potential risks while developing our project.

Safe Experiment Design

Our project was designed with safety precautions in mind to minimize risk at every stage of development.

Non-Pathogenic Chassis:

We used the E. coli strains DH5α and BL21. These non-pathogenic strains were chosen for their categorization as low risk for the environment and end user.

Safe Genetic Parts:

All protein-coding genes were selected based upon their status as non-hazardous and BSL-1 designation.

Inducing Autotrophy:

rhc_cbbL and rhc_cbbS: Derived from Rhodobacter capsulatus. Form I RuBisCO large and small subunits.

psp_rbcX: Derived from Picosynechococcus sp. RuBisCO assembly chaperone

csp_prkA: Derived from Cereibacter sphaeroides. Phosphoribulokinase is an enzyme in the Calvin cycle that regenerates one of the substrates for RuBisCo (the 5 carbon RuBP) by catalyzing the phosphorylation of Ru5P to RuBP using ATP

psp_FDH - Derived from Pseudomonas sp. NAD+-dependent Formate Dehydrogenase. Catalyzes oxidation of formate to CO₂ with reduction of NAD⁺ to NADH.

dci_bCA: Derived from Dolichospermum circinale. Encodes β-carbonic anhydrase; rapidly hydrates CO₂ ⇄ HCO₃^-.

Acetate Production:

ckI_pta: Derived from Clostridium kluyveri. Encodes Phosphotransacetylase which catalyzes the revisible conversion: acetyl-CoA ⇄ acetyl phosphate ⇄ acetate.

bsu_ackA: Derived from Bacillus subtilis. Encodes acetate kinase which catalyzes reversible interconversion of acetyl phosphate and ADP into acetate and ATP

PHBV Production:

hme_bktB: Derived from Haloferax mediterranei: β-ketothiolase; acetyl-CoA condensation

ssp_phaB: Derived from Synechocystis sp. Acetoacetyl-CoA reductase; reduces acetoacetyl CoA → 3HB-CoA.

avi_phaC: Derived from Allochromatium vinosum. This is a subunit of a class of polydroxyalkoanoates (PHA) synthase.

sfl_tdcB: Derived from Shigella Flexneri. converts L-threonine → 2-ketobutyrate + NH₄⁺ (anaerobic), feeding the HV branch (with TdcE + BktB) toward 3-ketovaleryl-CoA.

eco_tdcE: From Escherichia coli: 2-ketobutyrate formate-lyase catalyzes the conversion of 2-ketobutyrate to formate and propionyl-CoA, a necessary precursor for 3HV.

saz_aCA: Derived from Sulfurihydrogenibium azorense. rapidly hydrates CO₂ ⇄ HCO₃^-, elevating intracellular bicarbonate to activate soluble adenylyl cyclase (ADCY10), linking inorganic carbon to cAMP signaling and CRP-responsive promoters.

sfl_yciA: Derived from Shigella flexneri. An Acyl-CoA thioesterase, which in E. coli, is a major contributor to the overall thioesterase activity within the cell, and it hydrolyzes intermediates involved in both fatty acid synthesis and degradation.

yep_tesB: Derived from Yersinia pestis. Catalyzes the hydrolysis of a broad range of acyl-CoA esters to produce free fatty acids and CoA-SH. This will combat pyruvate buildup resulting from the knockdown of citrate synthase. While this enzyme is derived from a pathogenic organism, tesB is a conserved thioesterase amongst prokaryotes and is not recognized as a virulence factor; it produces benign metabolites, namely free fatty acids and CoA-SH.

Sensing and Reporters:

hsa_sACcat: Human soluble adenylyl cyclase; produces cAMP from ATP in response to HCO₃^-/Ca²⁺, independent of GPCRs, making it a direct CO₂/HCO₃^- sensor for coupling to CRP-responsive control.

msGFP2: Derived from Aequorea victoria Fluorescent reporter protein.

CRISPRi Module:

sadCas9: Derived from Staphylococcus aureus Encode a catalytically inactive dCas9, with a complementary sgRNA targeting E. coli gltA (Citrate syntase).

Non-Toxic Materials:

Whenever possible, we used safe, non-toxic reagents that met experimental goals without introducing undue hazards.

Lab Safety Procedures:

Safety of the team members was of utmost importance while conducting activities within the lab. Managing biobricks and wet-lab protocols required performing our due diligence and adhering to safety protocols.

Personal Protective Equipment:

Team members were required to wear appropriate PPE, consisting of a lab coat and gloves, to minimize risk of exposure to chemicals and biological agents. Prior to beginning lab work, all members completed intensive training on wet-lab safety and proper equipment use.

Chemical Usage:

All chemicals were handled with care, and the team was mindful of their associated risks and hazards while performing experiments.

Protocols for storage and disposal were taken from manufacturer-specific Safety Data Sheets (SDSs) and established before lab work began.

Sterile Technique:

Sterile technique was strictly maintained to prevent contamination when handling microorganisms. Bunsen burners were utilized to sterilize designated working zones.

• Liquid-contaminated samples were mixed with bleach to a contamination of 10%, then diluted with water and poured in the sink, unless other disposal procedures were designated by the manufacturer.
• Solid contaminants were autoclaved and disposed of in a designated waste container, unless other disposal procedures were designated by the manufacturer.

Waste Disposal:

All biological and chemical waste was disposed of in accordance with the University of Rochester’s Environmental Health and Safety department. Specific procedures were followed for autoclaved waste and liquid disposal. All reagents and waste products were clearly labeled.

Commitment to Safety

Throughout the project, safety was the highest priority. Our team ensured careful lab practices, minimized risks, and upheld the standards set forth by both the University of Rochester and iGEM.