Team:BABS UNSW Australia/Practices/Biosafety

Biosafety has been one of the major paradigms of project endosynbio. Our use of virulence factors and RG2 organisms, as well as the pathogenic nature of our engineered bacteria and the fact that this is our university’s first time at iGEM, has meant our intrinsic project design, protocol design and wet lab have been managed with extra regard for safety. See below for our final Safety Forms.

Something which has struck our team on this journey, however, has been the lack of regulation and guidelines, especially in Australia, regarding unforeseen consequences in synthetic biology. We believe a shift in the philosophy of bacterial genetic engineering could really help to improve this and as such, have explored this as a human practices endeavour. Please see our detailed report under the human practices menu, “Transforming biosafety: a new philosophy in synthetic biology.”

Final safety form

New parts

Part category	Species	Risk Group	Risk Group Source	Disease risk to humans Y/N	Part number and name	Natural function of part	How was this part acquired?	How will you use it?
pH promoters	Escherichia coli K12	1	Canada PSDS	N	Acid shock response promoter	Promotes the transcription of genes involved in the acid shock response	Synthesised in gBLOCK by IDT	To promote the expression of listeriolysin O in the acidic endosome
pH promoters	Lactococcus lactis subsp. lactis	1	Canada PSDS	N	P170 pH promoter	Involved in transcription of the cad operon	Synthesised in gBLOCK by IDT	To promote the expression of listeriolysin O in the acidic endosome
Invasion	Listeria monocytogenes	2	Canada PSDS	Y Listeriosis	Listeriolysin O	Virulence factor which permeabilises and ruptures the phagosomal membrane	Registry of Standard Parts Synthesised in gBLOCK by IDT	To allow bacteria to enter the mammalian cell cytoplasm
Invasion	Yersinia pseudotuberculosis	2	Canada PSDS	Y Acute enteric disease	Invasin	Binds to beta-1 integrins on mammalial cell surfaces to induce endocytosis	Registry of Standard Parts Synthesised in gBLOCK by IDT	To induce endocytosis of bacteria
Pseudoknots	Murine mammary tumour virus	2	American Biological Safety Association	N	MMTV pseudoknot	Diverse roles including frameshift device	Synthesised in gBLOCK by IDT	To delay transcription of downstream genes by a quantified time value
	Avian infectious bronchitis virus	2	American Biological Safety Association	N	IBV pseudoknot
	Human coronavirus 229E	2	Canada PSDS	Y Flu-like illness	HCoV 229E pseudoknot
	Porcine transmissable gastroenteritis virus	2	American Biological Safety Association	N	TGV pseudoknot
Toxin safety switch	Pseudomonas aeruginosa	2	Canada PSDS	Y Nosocomial infections including pneumonia	Tse2 toxin	Toxin	Registry of Standard Parts	To kill cells when they do not simultaneously possess the sister plasmid, carrying the anti-toxin
	Pseudomonas aeruginosa	2	Canada PSDS	Y Nosocomial infections including pneumonia	Tsi2 anti-toxin	Specific self-protection mechanism		To protect cells from the toxin
	P1 bacteriophage	N/A	N/A	N	Cre recombinase	Site-specific recombination		To excise invasin and listeriolysin once cytoplasmic invasion is achieved
	P1 bacteriophage	N/A	N/A	N	Lox sites	Site-specific recombination

Chassis Organisms

What is your chassis organism? Do you plan to experiment with any other organisms, besides your chassis?

Species name	Risk Group	Risk Group Source	Disease risk to humans Y/N	How was this organism acquired?	How will you use it?
Lactococcus lactis subsp. lactis	1	Canada PSDS	N	Internal source at UNSW (Jani O’Rourke lab)	These are our chassis organisms, or symbionts.
Escherichia coli K12 Dh5-alpha	1	Canada PSDS	N	Internal source at UNSW (Marc Wilkins lab)
Synechocystis PCC6803	1	NIH	N	Internal source at UNSW (Brett Neilan lab) and Macquarie University (Rob Lowe lab)
HeLa cells*	2	NIH	N	Internal source at UNSW	These are our host cells for infection with symbionts.
BHK-21 cells	2	NIH	N	Internal source at UNSW
CHO-7 cells	2	NIH	N	Internal source at UNSW (Andrew Brown lab)

* Carries lysogenic HPV DNA within genome, however believed to pose no risk to humans

How will your project work?

Our modified bacteria will be able to invade mammalian host cells using an IPTG and low pH driven invasin/listeriolysin O system (and in future work, hopefully be maintained at a stable population using AHL signalling systems). Our plasmid design ensures the invasive genes are de-activated once this invasion has occurred successfully, using delayed-timer pseudoknots and Cre recombination. A safety-switch system, maintained on two plasmids and integrated with the invasion system, will ensure no bacteria which leave the lab remain invasive and prevents horizontal gene transfer. This system is designed to pave the way for stable 'synthetic organelles' to be developed, with a medical application in mind.

Laboratory Risks

What risks does your project pose at the laboratory stage? What actions are you taking to reduce those risks?

Our project raises significant biosafety risks, as we are essentially generating novel pathogenic bacterial strains. In recognition of this, we assessed and managed our project laboratory risks using the Hierarchy of Hazard Control. The Hierarchy consists of five methods for hazard reduction beginning with the most effective, elimination, to the least effective, personal protective equipment. By using this system, risks can be managed appropriately. Some key examples of our risk management actions are illustrated below, as they were dealt with:

ELIMINATION

Many of our parts come from RG2 organisms, some of which are human pathogens. Instead of directly handling these organisms, we chose to order parts to be shipped from the Registry of Standard Parts or to be synthesised in the form of gBLOCKs from IDT. Thus this potential hazard was eliminated.

SUBSTITUTION

When ordered from the registry, we received an agar stab wherein invasin and lysteriolysin O were being constitutively expressed. This was extremely concerning to our team. Thus, the constitutive promoter was removed and replaced in all of our invasive constructs by the inducible IPTG promoter.

ENGINEERING CONTROLS

Within a PC1 lab, a Class II biosafety cabinet was used for all bacterial work both to protect our work from contamination and to protect ourselves, and other laboratory users, from the invasive bacteria. A timer was installed near the cabinet to ensure UV decontamination occurred for at least 20 minutes following a wipe-down with 70% ethanol. Periodically, the efficacy of the hood was checked by resting an uncovered nutrient agar plate inside the cabinet, before incubation overnight at 37 degrees.

All mammalian cell culture work occurred in a PC1 lab within a tissue culture hood.

ADMINISTRATIVE CONTROLS

All team members completed Workplace Health and Safety, Ergonomics, Laboratory Safety and Green (environmental awareness) Laboratory courses to update basic safety knowledge. We were inducted into all laboratories and common equipment rooms. All reagents were labelled appropriately as either HAZARDOUS or NON-HAZARDOUS and briefed with safety information, the date and the owner. A computer was established within the laboratory with access to a copy of the laboratory safety manual, chemical inventory lists, biological inventory lists, safe working practices (SWPs) and risk assessments. Materials safety data sheets were also directly accessible.

We are also researching and composing a report into the efficacy of current biosafety regulations, given the rapidly changing field and the lack of philosophical discussion regarding what it means to create a novel, persisting strain of bacteria. This work meant our team was in a safe, positive headspace regarding project decisions.

PERSONAL PROTECTIVE EQUIPMENT

Gloves and laboratory gowns were worn by all team members when in the lab.

Real World Applications

How would your project be used in the real world?

Our project is in the first instance foundational. Much more research is required to optimise and ensure the safety and durability of this system. However, our long-term vision is to see these synthetic organelles be used in the human body. With the rise of organogenesis, this may be possible in the near future.

Product Risks

What risks would your project pose, if it were fully developed into a real product that real people could use? What future work might you do to reduce those risks?

The main risk would be the organisms becoming uncontrolled and invading non-target cells. Future work would require adding invasion specificity and extensive testing of how well the toxin/anti-toxin system functions. Another risk could be horizontal gene transfer of the toxin plasmid, causing damage to normal cells. We recognise that this kind of technology would be very difficult to trial in vivo.