Biosafety and Biosecurity

Biosafety and Biosecurity

Our venture into biosafety and biosecurity began quite early in the ideation cycle, but made its first significant strides after the iGEM ambassadors’ biosafety, biosecurity, and dual-use research concerns (DURC) workshop at the All-India iGEM Meet (AIIM).

According to the USA National Research Council, biosafety is about protecting people from ‘bad bugs’, and biosecurity is about protecting bugs from ‘bad people’. To put it more formally, biosafety is concerned with preventing accidental exposure to and release of biological materials, while biosecurity deals with preventing the intentional and malicious misuse of biological materials.

Any synthetic biology project has the possibility of falling victim to either or both situations and facing the consequences thereof. Therefore, we have taken concrete steps towards ensuring we cover the predictable dark paths our project might venture into.

Laboratory Safety and Preparation

The lab training we have received as part of our curriculum will enable us to conduct ourselves and our experiments with maximum care and discipline. The lab space we have access to is a fully functional biology laboratory, with sterilization equipment, PPE, safety showers, fume hoods, and laminar air-flow chambers.

The Office of Laboratory Safety and Environmental Health (OLSEH) is the governing body concerned with laboratory risk management and mitigation at IISc. They have pre-set Incident and Investigation report forms, and also handle laboratory waste disposal for the Institute. Furthermore, as part of our Institute’s policy, all our wet lab team members have taken the online OLSEH safety test to ensure readiness to work inside the laboratory. The training material relevant to us in the safety test covers the following modules:

• General Safety: This module is mandatory for all.
• Chemical Safety: Required for all those who plan to work with chemicals (NFPA rating > 1).
• Biological Safety: Required for all who plan to work in bio-labs.

Finally, waste disposal at our lab is handled by experts at OLSEH, with properly segregated disposal systems for different materials.

Kill Switch

Experimentally, we do not have an engineered bacterial kill switch in the conventional sense of the word. Instead, the genes we insert into the bacterium will only get expressed to form nitrite-reducing enzymes when in the presence of sufficient concentrations of benzoate (a plant root exudate). We have done this by using the XylS/Pm inducible promoter-containing plasmid. This ensures that the bacterium only expresses the genes when it is present in the intended environment, which is a biofilm around plant roots. When the bacteria are not in the presence of benzoate—meaning when they are not around plant roots—they will not metabolize nitrate.

Theoretically, they may spread uncontained while having the exact genetic makeup of wild-type Pseudomonas putida KT2440. Since P. putida KT2440 is a well-studied native soil bacterium, mutation permitting, the environment should not be harmed even with its spread.

This, despite not being a kill switch in the conventional sense, will not allow the modified bacteria to proliferate in an environment where it is not supposed to. We understand that this might not be an effective strategy when translated outside the laboratory, particularly when we consider environmental deployment.

With the increasing use of genetically modified organisms (GMOs) over a wide range of applications arise some major ecological and ethical questions surrounding the impact these organisms may possess towards their surroundings. In the project, use of modified strains of Pseudomonas putida brings to forefront some issues which may arise as a result of the strain escaping out of its ‘zone of containment’. For our system in particular, this zone of containment can be defined as belonging to the roots of paddy, wheat etc crops where Pseudomonas putida colonies exist in the form of biofilms.

Bacteria especially, showcase many abilities which makes them very hard to deal with once modified strains escape. Since they are capable of horizontal gene transfer, the recombinant DNA of its engineered genes can be transferred to any wild microbial group which can lead to some unpredictable long-term consequences among the bacterial populations in the region. This is exacerbated by the selective pressures in these diverse natural habitats might favour these modified traits if they offer even a slight competitive advantage. Other than the ecological threat it poses, such cases of leak/escape raise ethical issues among the public.

So, as a result it is extremely important to limit the spread of any GMO microbes outside their intended conditions. This is achieved by biocontainment systems. Biocontainment systems fundamentally aim to achieve confinement of these microbial GMOs by engineering various safeguards into the strains which relate microbial viability to the changes in external conditions which the microbe might face during escape. Various methods to do the same have arisen where the genetic pathways in the microbes are either modified or added to tie its survival to certain external factors.

The oldest developed among these is metabolic auxotrophy, where the whole genome of the microbe is modified to link its survival to the presence of any exogenously supplied metabolite. Since, the microbe cannot produce the metabolite on its own and their survival is so intrinsically intertwined to it, on removal of the supply, death would occur. For this project, the common root metabolite of such plants, benzoate will play a central role, acting as an important internal signal, the absence of which will form the basis of many a type of biocontainment genetic method.

But this method also comes with some caveats. If the metabolite is somehow supplied to the bacteria in external environment, e.g.: through crossfeeding via other microbes or due to changing conc. of metabolites in different areas (which always carries some possibility), the biocontainment strategy retains chances of failure. This method also relies on widespread changes in the genetic code, creating an impractical system where modulation and modification is extremely hard.

Due to these factors, there was need for a system which is more customisable where more the survival is linked not to the overall metabolic system, but to a small genetic circuit engineered into the microbe, the gene expression of which are controlled by some containment signal from the surroundings. As their expression can be turned ‘ON’ and ‘OFF’ by the signals, these genetic devices are called kill switches. These signals can either maintain essential gene expression or block toxin production. Once signal is removed, the reverse occurs, and cell death is triggered by toxin production or termination of essential gene expression.

The signals used are a matter of wide selectivity, a variety of different inputs are accepted and for greater selectivity, a combination of multiple different ones can be used. Even if regulatory logic changes with change in external signals, the overall role of its functionality remains the same. They are thus, very programmable and find easier applications on a greater variety of cases. Kill switches are of multiple different times, each employing their own specific style of genetic circuit which relates with the signals to overall lead to similar ends towards biocontainment.

The Deadman variety of kill switches work by relating constant input of signal with survival. The input can be associated with expression of essential functions. Or, once when the input stops, strong expression of toxic gene can lead to death of the cell (possibly explained by the lack of expression of toxin repressor). Such a theoretical circuit for the system of crop plant roots where P. putida form biofilms would relate essential gene expression to a common exudate compounds of the rhizosphere like benzoate and its derivatives. Classically, these are toxin-antitoxin systems.

The input signals can be monitored using a benzoate-responsive regulator/promoter pair (examples: BenR → Pb, or XylS → Pm). These act as the sensors that report presence or absence of the root exudate and lead to the follow up expression.

In theory, this is how the survival controls work:

More specifically,

• Positive Survival Control Deadman:
  • IF benzoate present (the sensors BenR/XylS active) → promoter ON → essential function expressed → cell lives.
  • ELSE no benzoate → promoter OFF → essential function absent → cell cannot survive.

• Toxin Repression Control Deadman:
  • IF benzoate present → sensor activates expression of a repressor protein that suppresses toxin expression → toxin OFF → cell lives.
  • IF benzoate absent → repressor not expressed → toxin freely expressed (ON) → cell dies.

• The possible sensors chosen are BenR/Pb, a natural benzoate sensor, and XylS/Pm, which responds to methyl or substituted benzoates.
• To further increase specificity, another type called the Passcode kill switch can be utilized where only a specific combination of multiple inputs can give rise to a repressor to a toxin whose expression is otherwise always incorporated. Other than exudate molecules acting as inducers for sensors, other external physicochemical conditions like O₂ levels, surrounding pH, etc. can be related to survival for greater specificity in containment action. In the absence of all the requisite factors, it stops repressor production leading to death by toxin expression. This system might prove relevant in the highly homogenized settings of agricultural fields where much of the soil sites of action retain similar physicochemical conditions while those outside change sharply. As a result, uncontrolled external spread from agricultural land is prevented. Horizontal gene transfer outside is also prevented from becoming commonplace by the lethality of the systems involved.
• Genetic stability challenge:

  But there is one major weakness associated with biocontainment systems like these in general which is their genetic stability or the lack thereof. As we create a circuit which reduces the fitness of the bacteria involved, a strong selection pressure against these strains is imminent. As with any genome, many mutations will arise. And any mutations which lead to a loss of function (with respect to the genetic circuit) showcase much higher fitness than the original modified strains and hence, they proliferate. The cost associated with reduced fitness is felt even within the root systems and hence the containment strategy is selected against for in the root itself. Over a surprisingly short period of time (few days even) the bacterial population seems to by and large have lost all the containment related circuits incorporated in them. To prevent this, a different strategy needs to be incorporated so that the kill switches are genetically stable.

• The Demon & Angel concept: A single-construct approach where both the survival and death of the bacteria is tied to the expression of the same gene which shows different effects at different levels of its expression. The levels of expression of the gene can be tied with external signals which are received by bacteria.
  To incorporate into such a circuit, we select such a gene which is essential at low levels of its expression but becomes toxic when overexpressed. At low expression (Angel state), the gene is essential and helps maintain the cell’s viability and lets it continue its normal existence. But at high expression (Demon), the same gene overexpressing leads to toxin accumulation and killing of the host cell. This linking of both viability and killing of the host cell with the same gene is what is the principle behind this acting like a genetically stable system. Any loss of function mutation here, would inactivate not only the toxin, but also inactivate the low-level expression required for the survival of the microbe itself. So, such a mutant would instead be selected against and the kill switch’s genetic stability over generations is seen to be maintained.

The mechanism behind such a circuit can be explained by the following:

• Low Gene Expression (OFF State):
  • When the bacterium is in the rhizosphere or attached to the plant root, the intended deployment area, the cell detects a high concentration of specific root exudates released by the plant roots which act as our signals here. The presence of the signal keeps the kill switch in the OFF state.
  • A small molecule-binding transcription factor (TF) senses the root exudate, and its binding represses the overexpression of the toxic gene or induces the expression of a protective anti-toxin/inactivator gene. This allows the essential gene and thus, the entire cell to function normally.
• High Gene Expression (ON State):
  • When the bacterium escapes the root system and moves into the external environment, the cell experiences a loss of the specific root exudate signal. The loss of signal switches kill switch to its ON state, i.e. towards its overexpression.
  • The transcription factor no longer binds the root exudate/signal, causing it to stop repressing the toxic gene, leading to high, lethal expression. The resulting overexpressed essential gene product kills the cell, thus containing the engineered microbe in its environment.

More specifically:

• Sensor Module: Helps relate the demon/angel component with the external signal which determines the level of expression of the gene. It uses the same benzoate sensors to detect rhizosphere specific metabolites, BenR → Pb or XylS → Pm like before. The sensor module of the circuit controls both the demon and angel components of the circuit indirectly.
• Angel Module (Pro-survival):
  • If benzoate present → Angel ON → essential gene expressed → cell survives.
  • If benzoate absent → Angel OFF → essential function not expressed → cell weakened, preparing Demon activation.
• Demon Module:
  • Demon is normally OFF (repressed) when Angel is active.
  • If Angel activity stops (benzoate absent, or Angel is mutated) → Demon activated → gene overexpressed, becomes toxic to cell → cell dies.

Now, the specific gene which is toxic on overexpression but essential in lower amounts is something which needs to be selected to prepare the circuit in question. A possible example of such a gene system might be:

• ftsZ under control of XylS/Pm (Hypothetical):

  The ftsZ gene encodes a cytoskeletal protein that is the prokaryotic analogue of tubulin and plays a key role in cytokinesis during bacterial cell division. It forms the contractile structure known as the Z-ring at the future site of cell division.

  In the rhizosphere, in the presence of benzoate, controlled expression of ftsZ ensures proper, balanced cell division. However, in the absence of benzoate, overexpression of the protein occurs, leading to mislocalized Z-rings and filamentation defects, which can be lethal to the bacteria.