Team:TU Dresden/Safety
Safety expertise
Risk assessment
Risk assessment of the Biosensor encapsulated in Peptidosomes
The membrane of Peptidosomes consists of Fmoc-FF molecules (diphenylalanine with a protection group) that self-assemble into net-like structures around the non-assembled core of the Peptidosome. Diffusive mass transport enables an exchange between the surrounding medium and the unassembled FmocFF solution in the center of the Peptidosome. Peptidosomes without encapsulated bacteria, do not hold genetically modified organisms (GMOs) and therefore do not fall under the corresponding acts/laws.
Peptidosomes will serve as a novel immobilization matrix for Bacteria and other microorganisms. Below, we describe the possibility of a potential utilization of the biosensor encapsulated into Peptidosomes in scientific research and industry. Hereby, the judicial evaluation will be based on the law on genetic engineering.
The law on genetic engineering underlies the so-called step by step principle, meaning the containment of GMOs is loosened gradually (vgl. Freisetzungsrichtlinie 2001/18/EG (24)). It is important to mention, that there has been no normative regulation set, which implies the processing of all steps is mandatory for approval. Here, the process is divided into three separate steps:
- Genetic engineering operations in a genetic engineering facility
- Release
- Placing on the market/Putting into circulation
All steps have been derived from the risk assessment of the GMOs, which is carried out dispositive after Anhang II der Freisetzungsrichtlinie 2001/18/EG as well as the decision 2002/623/EG.
This risk assessment features several stages:
- Investigation of characteristics, that potentially result in harmful effects
- Assessment of possible consequences of these harmful effects, if they occur
- Assessing the probability of the occurrence of the individual potential harmful effects
In the case of our biosensor, all genetic constructs that result in the biosensor activity are stably integrated into the genome of Bacillus subtilis W168. The classification of this bacterium into the risk group was extracted from the technical rules for biological working materials (Technischen Regeln für Biologische Arbeitsstoffe (TRBA 466)). This source rates Bacillus subtilis subsp. subtilis among the risk group 1 (R1), with the remark, that this bacterium was proved or rumored to have a pathogenic effect in rare individual cases, especially for people with a severely suppressed immune system. Often, the identification of this species as a pathogen was not reliable.
The genetic constructs constituting the biosensor were derived from the organism Staphylococcus aureus N135. In the TRBA 466 this organism is counted to risk group 2 (R2), with the remark, that this organism represents a pathogen to humans and vertebrates, but that there is no transmission between the two host groups possible. Additionally, our biosensor contains an operon from Photorhabdus luminescens subsp. Luminescens, which after TRBA 466 belongs to risk group 1 (R1), but can be a harmful pathogen for several invertebrate species.
The security classification of the biosensor is based on the consideration of the following aspects:
- Identifying all features of the utilized microorganism relevant for the overall safety
- Characteristics of the activity
- Severity and probability of a potential endangerment of protected goods of § 1 Nr. 1 GenTG
Bacillus subtilis, belonging to R1 represents no risk for humans and the environment, while the pathogenic R2 organism Staphylococcus aureus illustrates a potential hazard.
To build the biosensor, we selected two genes from S. aureus N315, blaR1 and blaI, which have been codon optimized for expression in B. subtilis and thus were chemically synthesized. The company (IDT DNA Technologies) responsible for the chemical synthesis of these genes has the responsibility of preventing the delivery of potentially pathogenic or virulent genetic sequences derived from S2 organisms to S1 laboratories. Furthermore, there is no connection of the used genetic sequences to any of the organism`s pathogenic mechanisms.
The first gene, blaR1, encodes a receptor for beta-lactam antibiotics. This receptor enables B. subtilis to sense compounds of this family in its surroundings, but not to degrade them. The second gene, blaI, is a repressor of transcription. This repressor inhibits gene expression of the operon luxABCDE serving as a reporter that will lead the cells to produce a luminescent signal in the presence of beta-lactam antibiotics. When the antibiotic compounds are absent, there will be no luminescent signal detectable. The operon luxABCDE originates from luminescens subsp. Luminescens. This microorganism is non-pathogenic for humans.
Further a mutation has been introduced to the genome of W168, by exchanging the gene penP with a resistance cassette for kanamycin, causing a deletion of large parts of its coding sequence. This gene normally codes for a beta-lactamase, making Bacillus more resistant to several beta-lactam antibiotics. We deleted this gene to make the biosensor more sensitive to the antibiotic compounds and to ensure that the compounds of interest are not degraded by our strain.
In summary, due to the introduction of the genetic constructs, the biosensor features resistant cassettes that facilitate resistance against Chloramphenicol (5mg/mL), MLS (1mg/mL Erythromycin; 25mg/mL Lincomycin), Kanamycin ((10mg/ml) and Spectinomycin (200mg/mL).
Accorrding to § 1 Nr. 1 GenTG (Genetic engineering act) life and health of mankind, the environment and its interacting systems, fauna, flora and material assets must be protected from any harmful effects of the biosensor. Due to the detailed characterization of the genetically engineered organism, it is grouped into safety level 1 and therefore constitutes no risk. In the case of genetic engineering operations in a genetic engineering facility, a notification is necessary. This security assessment is supported by the required elements after § 7 Abs. 2 Nr. 1 GenTSV for the production section and § 7 Abs. 3 Nr. 1 GenTSV.
As there is no normative securement/fixation of the principle for the passaging of the individual steps in the step by step principle, we hereafter focused directly on 3) putting into circulation, as the judicial question, in how far the application of the biosensor illustrates a release, could not be clarified.
The release underlies an authorization requirement. Condition for an authorization is set by § 16 Abs. 2 GenTG. Thus, an authorization can just be granted, when after the current state of science in relation to the purpose of placing it on the market, no unjustifiable harmful effects on the legal interests, as mentioned in § 1 Nr. 1 GenTG, are being expected. To determine, whether those conditions are fulfilled in this specific case, the purpose of release and the criteria for justification will be differenced in detail.
Purpose of release:
- Circle of users
- Object of users
- Precise application purpose
- Predicted value
Criteria for justification:
- Degree of probability of occurrence of harmful events
- Dimension of potential harmful applications
- Advantages of the intended application
- Substitutability with other products or methods
The application of the beta-lactam biosensor is not limited to one specific user. Due to the broad detection range, an application in a variety of sectors can be imagined. Hereby, the biosensor could potentially sense about 100 different compounds of the beta-lactam family, which are detectable even in very low concentrations (ng). Possible users are for example sewage treatment plants, food analysis laboratories (testing of meat) and other analytical laboratories in the field of antibiotic detection.
- Sewage treatment plants: The biosensor could be applied here to prove a potential contamination of the waste water with antibiotics.
- Food analysis: Proving the presence of antibiotics in meat samples is necessary, especially for livestock. Animals are typically treated with antibiotics and traces of the compounds could still be detectable in the meat.
- Analytical laboratories: With the biosensor, soil samples, as well as many other sample types can be examined to identify novel antibiotic compounds that feature similar chemical structures as the already characterized beta-lactam antibiotics.
Following, the biosensor is considered in the novel immobilization system “Peptidosome”. Especially the impact Peptidosomes have on the degree of the probability of occurrence of harmful effects has to be determined.
The release is justified particularly by the encapsulation of the biosensor into Peptidosomes as an additional safety precaution. Due to this permeable cage, the diffusive mass transport of liquids (medium) and small molecules is possible, but the encapsulated bacteria are held back on the inside. Therefore, an additional barrier is created between the GMOs and their environment.
In the case of the use in sewage treatment plants, various possible applications of the encapsulated biosensor can be imagined. There is the possibility to take water samples and transfer these into an S1-facility in which the test for antibiotic contamination using the biosensor is performed. Otherwise, we could think of an integration of the biosensor into the sewage treatment plant. Integration into a treatment plant would require reconstruction or renovations to the facility and demand a higher personnel requirement (compare to § 16 Nr. 1 GenTSV).
The GMOs must be prevented from escaping the S1-facility of the sewage treatment plant at any time. To do so, a system needs to be installed that can guarantee that no living GMO leaves the facility. In contrast to the use of the biosensor without any matrix for encapsulation, Peptidosomes serve as an additional envelope or shell that keeps bacteria from breaking out of their cage. Due to this barrier, the maintenance of the GMOs in the S1-facility can be guaranteed.
Furthermore, another visionary application in this sector constitutes a portable unit, which contains the encapsulated biosensor. This would enable the easy and fast examination of water samples in the field. This vision, in regard to application b), depicts a profitable improvement, precisely due to the easy transport of the biosensor and the low probability of occurrence of harmful effects. The extent of potentially harmful effects is marginal or even negligible. The construct of the biosensor represents an R1/S1-organism, which is not pathogenic for humans or harmful for the environment.
In summary, it can be stated that the genetic engineering assessment of the Peptidosome as immobilization matrix depends solely on the encapsulated bacteria. The Peptidosome is a further safety precaution against the release of the microorganisms into the environment.
