Contribution

During our project, our team encountered several challenges that made executing our project more difficult. Since other iGEM teams might run into similar challenges, we are making our developed tools available. First, we established that within our team little was known about the exact laws that apply to the European Union (EU) surrounding the use of genetically modified organisms (GMOs) in the environment. Also, when we tried to delve deeper into this, we found ourselves having to trudge through endless pages of detailed regulations. In order to make these regulations more insightful for us and future iGEM teams, we have created a regulatory roadmap. This regulatory roadmap serves as an outline for both (i) the different legislations that restrict the non-contained applications of GMOs and (ii) the conditions that have to be taken into account when applying for a license. Secondly, we have encountered that it is hard to compare the transcriptional activities of promoters from the iGEM registry. They were characterized using different equipment, different strains and within different gene constructs. Therefore, we developed a robust method to recalibrate inducible promoters, such as pBAD (BBa_I0500), using constitutive promoters. Our results add essential information on the use of pBAD in the iGEM registry.

Regulations surrounding the non-contained application of genetically modified organisms (GMOs) in the European Union

A legal framework for the possibilities and conditions for the deliberate release of GMOs in the environment

Introduction

This summary serves as an outline of the general laws and conditions concerning the non-contained application of genetically modified organisms (GMOs) inside the European Union. These regulations are described in Directive 2001/18/EC, which forms the basis for the legislation throughout member states of the EU. A directive is a general disquisition, which is aimed to represent the legislations surrounding a goal that is enforced in the member states throughout the EU.

With this analysis, we aim to provide an outline of the laws and conditions that must be taken into account when developing a non-contained GMO application in the EU. This outline aims to better understand the current possibilities and restraints concerning these non-contained GMO applications. Furthermore, this outline also provides a framework for the different aspects of biosafety that have to be taken into account by researchers developing solutions that need the non-contained application of a GMO. For these reasons, we believe that other iGEM teams aiming to apply GMOs in a non-contained setting could use this outline to easily identify the different restrictions their project is subject to and how the team can work towards realizing their solution.

Definitions of GMO and non-contained use
Before we dive deep into the regulations, we must first explain the definitions of some important terms according to the directive.
1. Genetically modified organism (GMO): an organism, with the exception of human beings, in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination. Thereby not including organisms that result from mutagenesis;
2. Deliberate release: any intentional introduction into the environment of a GMO or a combination of GMOs for which no specific containment measures are used to limit their contact with and to provide a high level of safety for the general population and the environment;
3. Environmental risk assessment: the evaluation of risks to human health and the environment, whether direct or indirect, immediate or delayed, which the deliberate release or the placing on the market of GMOs may pose;
4. Placing on the market: making available to third parties, whether in return for payment or free of charge;
5. Product: a preparation consisting of, or containing, a GMO or a combination of GMOs, which is placed on the market.

The core of the directive

The objective of this directive is to protect human health and the environment from possible hazards that arise with the release of GMOs. Furthermore, this directive provides the directions for the application of a permit for the use of these GMOs. Central to this directive is the environmental risk assessment (ERA). This ERA is designed to evaluate the potential risks of the deliberate release of GMOs to human health and the environment. The content of this ERA includes a step-by-step identification of the possible adverse effects that could arise with the evaluated GMO in its environment, as detailed in annex II of the Directive 2001/18/EC. With the results of the ERA, conclusions are drawn based on the risks and the corresponding safety measures for risk management. This ERA is important for the directive since it serves as a way to evaluate both the risks and the safety of the entire process.

License application
With the license application, all the information is provided for the assessment of the safety and environmental risks that remain with the introduction of the GMO. The required information includes:
1. The specified information required for the ERA (annex IIIA)
2. Content of the ERA (annex IIC)
3. Conclusions of the potential environmental impact from the release of GMOs (annex IID)
With this information, a license application can be submitted to the responsible regulatory institution. Each European country is monitored by its own institution, for example, Rijksinstituut voor Volksgezondheid en Milieu (RIVM) in the Netherlands.

The specified information required for the ERA

Implications: As part of the license application, information must be provided that serves as the foundation for the execution of the ERA. This information describes the characteristics of the GMO, the environmental surroundings of the potential release, and the suggested control of the potential consequences. This is important to gather and showcase the information that is consequently used to perform the ERA. This required information includes (annex IIIA):
1. General information including information on personnel and training
2. Information relating to the GMO(s)
  1. Characteristics of the donor, recipient, parental organism(s), among others taxonomy, description of the geographic distribution and pathological, ecological, and physiological traits.
  2. Characteristics of the vector: nature and source, among others.
  3. Characteristics of the modified organism, among others information relating to the modification and the final GMO.
3. Information relating to the conditions of release and the potential receiving environment
  1. Information on the release
  2. Information on the environment
4. Information on the interactions between the GMO(s) and the environment
  1. Characteristics affecting survival, multiplication, and dissemination
  2. Interactions with the environment
5. Information on monitoring, control, waste treatment, and emergency response plans
  1. Monitoring techniques
  2. Control of the release
  3. Waste treatment
  4. Emergency response plans
6. A plan for monitoring in accordance with the relevant parts of Annex III to identify effects of the GMO(s) on human health or the environment
7. Information on control, remediation methods, waste treatment and emergency response plans
8. A summary of the dossier
Content of the ERA

Implications: As previously stated, the ERA is a fundamental part of the license application and serves as a way to identify possible hazards that come along with the intended release of the GMO. This is important for the applicants to evaluate the risk that is posed with the proposed methods and the sufficiency of the intended safety measures. The execution of this ERA is based on a step-by-step assessment of these possible hazards and includes the assessment of the points:
1. Identification of characteristics that may cause adverse effects, e.g. disease to humans, animals, or plants.
2. Evaluation of the potential consequences of adverse effects, if it occurs
3. Evaluation of the likelihood of the occurrence of each identified adverse effect
4. Estimation of the risk posed by each identified characteristic of the GMO
5. Application of management strategies for risks from the deliberate release or marketing of GMO(s)
6. Determination of the overall risk of the GMO(s)
Conclusions of the potential environmental impact from the release of GMOs

Implications: The last aspect of the license application includes the formulation of the conclusions of the ERA. This part is included to assist in formulating conclusions on the potential environmental impact from the release or the placing on the market of GMOs. This, therefore, serves as the final establishment of the possible risks to the environment that could be posed by the introduction of GMOs. To draw this conclusion, the following points should be addressed:
1. Likelihood of the GMO to become persistent and invasive in natural habitats under the conditions of the proposed release(s).
2. Any selective advantage or disadvantage conferred to the GMO and the likelihood of this becoming realized under the conditions of the proposed release(s).
3. Potential for gene transfer to other species under conditions of the proposed release of the GMO and any selective advantage or disadvantage conferred to those species.
4. Potential immediate and/or delayed environmental impact of the direct and indirect interactions between the GMO and target organisms (if applicable).
5. Potential immediate and/or delayed environmental impact of the direct and indirect interactions between the GMO with non-target organisms, including impact on population levels of competitors, prey, hosts, symbionts, predators, parasites, and pathogens.
6. Possible immediate and/or delayed effects on human health resulting from potential direct and indirect interactions of the GMO and persons working with, coming into contact with or in the vicinity of the GMO release(s).
7. Possible immediate and/or delayed effects on animal health and consequences for the feed/food chain resulting from consumption of the GMO and any product derived from it, if it is intended to be used as animal feed.
8. Possible immediate and/or delayed effects on biogeochemical processes resulting from potential direct and indirect interactions of the GMO and target and non-target organisms in the vicinity of the GMO release(s).
9. Possible immediate and/or delayed, direct, and indirect environmental impacts of the specific techniques used for the management of the GMO where these are different from those used for non-GMOs

Conclusions
To summarize this analysis:
1. The application of non-contained GMOs is possible throughout the European Union, following a license application and allocation from the responsible regulatory institution.
2. For each application, an environmental risk assessment (ERA) has to be performed, to evaluate the potential risks of the release to human health and the environment.
3. The final license application has to include: the specified information for the execution of the ERA, the ERA, and the conclusions from the ERA.

References

DIRECTIVE 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC. (OJ L 106, 17.4.2001, p. 1) http://eur-lex.europa.eu/legal-content/EN/TXT/?qid=1375683320071&uri=CELEX:32001L0018

Promoter Calibration

Introduction

The DOPL LOCK system is based on toxin-antitoxin systems (TA systems) to prevent horizontal gene transfer and physically containing genetically modified organisms. The expression of the toxin-antitoxin system requires strict regulation to maintain the viability of the engineering strains and the functions of DOPL LOCK. However, we encountered difficulties in deciding which promoters should be used to maintain a balanced toxin-antitoxin ratio. We also found that the provided information on the inducible promoter pBAD (BBa_I0500) in the iGEM registry was not sufficient to design balanced TA systems. Therefore, we have developed a method to calibrate pBAD with constitutive promoter p2547 (BBa_J23100) in Escherichia coli TOP10. The method can be effortlessly implemented for future iGEM teams to calibrate other promoters. Furthermore, our results conclude the transcriptional activities of pBAD and its relative activity to p2547 at different L-arabinose concentrations. It adds essential information on the use of pBAD in the iGEM registry and to future iGEM teams who would need tight regulation on their genetic circuits.

Calibration of pBAD

Method

We first cloned promoters in front of the gene of the fluorescent protein mCherry (BBa_K3962339 and BBa_K3962340) and transformed the constructs into E. coli TOP10. The fluorescence of the strains was measured by the plate reader when their OD₆₀₀ reached 0.5. The measurement lasted 10 h with time intervals of 5 minutes. Then, their fluorescent intensities were fitted to a standard form of the logistic equation and the maximum values of the models were calculated. The relative fluorescence and the logarithm of arabinose concentration were finally fitted into a linear regression.

More detailed information can be found at Measurement Page

Results

The result showed constant expression from p2547::mCherry and the expression from pBAD::mCherry increased gradually as the concentration of arabinose increased (Figure 1). The lowest fluorescent protein expression was observed at the concentration of 0.00032% (w/v). We also concluded that pBAD is tightly regulated as it showed low leaky expression when no arabinose was added.

Figure 1: The fluorescence intensity of strains carrying the mCherry constructs under different arabinose concentration. The measurements were carried out for 20h. Blue lines and dots were fluorescence of p2547 while red ones originated from pBAD. The arabinose concentration was shown at the bottom of each graph.

The relation between the arabinose concentration (w/v, log₅) and the relative fluorescence of pBAD fitted into a linear regression model with R² = 0.9586 (Figure 2). From the regression it could be calculated that at the arabinose concentration of 0.14%, the transcriptional activity of pBAD was equal to p2547.

Figure 2: Linear regression of arabinose concentration relative fluorescence of pBAD compared with p2547. The orange dash lines showed how p2547 related to pBAD transcriptional activity at the arabinose concentration of 0.14% (log₅-1.22).

Detailed information about this part can be found on the Measurement page

Compared to former calibration, our calibration method of pBAD included an internal reference of fluorescent intensity by testing the constitutive promoter in the same biological environment. Using p2547 as the reference, the deviation of fluorescence caused by different strain, different growth conditions and different measuring equipment can be minimized. Moreover, previous quantitative calibration only collects fluorescence data at one time point and uses them as the response. Our measurement time includes the lag phase, exponential phase and the stationary phase of the bacteria growth. Therefore, the fluorescence curve can be fitted to a logistic function and the maximum fluorescence can be used to quantify the transcriptional activity. We have uploaded our results to the iGEM registry for the future iGEM teams to design their genetic circuits.

Team:Leiden/Contribution

Contribution

Regulations surrounding the non-contained application of genetically modified organisms (GMOs) in the European Union

A legal framework for the possibilities and conditions for the deliberate release of GMOs in the environment

Introduction

Definitions of GMO and non-contained use

The core of the directive

License application

The specified information required for the ERA

Content of the ERA

Conclusions of the potential environmental impact from the release of GMOs

Conclusions

References

Promoter Calibration

Introduction

Calibration of pBAD

Method

Results

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