1) Biosafety

Biosafety is defined as the real or potential danger to human beings, animals or plants by direct infection or indirect destruction of the environment by biological infectious agents.

Biohazard

The term "laboratory biosafety" is used to describe the protective principles, techniques and practices used to prevent accidental exposure and accidental release of pathogens or toxins.

a) Laboratory safety classification

The term "laboratory biosafety" is used to describe the protective principles, techniques and practices used to prevent accidental exposure and accidental release of pathogens or toxins.

b) Engineering bacterial classification

The backbone of the practice of biosafety is risk assessment. One of the most helpful tools available for performing a microbiological risk assessment is the listing of risk groups for microbiological agents. However, simple reference to the risk grouping for a particular agent is insufficient in the conduct of a risk assessment. Other factors that should be considered, as appropriate, include:

1. Pathogenicity of the agent and infectious dose;
2. Potential outcome of exposure;
3. Natural route of infection;
4. Other routes of infection, resulting from laboratory manipulations (parenteral,airborne, ingestion);
5. Stability of the agent in the environment;
6. Concentration of the agent and volume of concentrated material to be manipulated;
7. Presence of a suitable host (human or animal;
8. Information available from animal studies and reports of laboratory-acquired infections or clinical reports;
9. Laboratory activity planned (sonication, aerosolization, centrifugation, etc);
10. Any genetic manipulation of the organism that may extend the host range of the agent or alter the agent’s sensitivity to known, effective treatment regimens; (Staff & Organization, 2004) (Staff & Organization, 2004) Staff, W. H. O., & Organization, W. H. (2004). Laboratory biosafety manual: World Health Organization. .

c) Biosafety considerations for expression vectors

Higher biosafety levels may be required when: 1. The expression of DNA sequences derived from pathogenic organisms may increase the virulence of the GMO;
2. Inserted DNA sequences are not well characterized, e.g. during preparation of genomic DNA libraries from pathogenic microorganisms;
3. Gene products have potential pharmacological activity;
4. Gene products code for toxins;

c) Engineering bacteria

Recombinant DNA technology involves combining genetic material from different sources thereby creating genetically modified organisms (GMOs) that may have never existed in nature before. Experiments involving the construction or use of GMOs should be conducted after performing a biosafety risk assessment. The properties of the donor organism, the nature of the DNA sequences that will be transferred, the properties of the recipient organism, and the properties of the environment should be evaluated. These factors should help determine the biosafety level that is required for the safe handling of the resulting GMO, and identify the biological and physical containment systems that should be used (Berg, Baltimore, Brenner, Roblin, & Singer, 1975) (Berg, Baltimore, Brenner, Roblin, & Singer, 1975) Berg, P., Baltimore, D., Brenner, S., Roblin, R. O., & Singer, M. F. (1975). Asilomar conference on recombinant DNA molecules. Science, 188(4192), 991-994. .

What about biological expression systems?

Biological expression systems consist of vectors and host cells. A number of criteria must be satisfied to make them effective and safe to use. An example of such a biological expression system is plasmid pUC18. Frequently used as a cloning vector in combination with Escherichia coli K12 cells, the pUC18 plasmid has been entirely sequenced. All genes required for expression in other bacteria have been deleted from its precursor plasmid pBR322. E. coli K12 is a non-pathogenic strain that cannot permanently colonize the gut of healthy humans or animals. Routine genetic engineering experiments can safely be performed in E. coli K12/pUC18 at Biosafety Level 1, provided the inserted foreign DNA expression products do not require higher biosafety levels (Directive, 1990) (Directive, 1990) Directive, C. (1990). Council Directive 90/219/EEC of 23 April 1990 on the contained use of genetically modified micro-organisms. Official Journal L, 117(08/05), 0001-0014. .

2) Hazards arising directly from the inserted gene

Assessment is necessary in situations where the product of the inserted gene has known biologically or pharmacologically active properties that may give rise to harm, for example:
1. Toxins;
2. Cytokines;
3. Hormones;
4. Gene expression regulators;
5. Virulence factors or enhancers;
6. Oncogenic gene sequences;
7. Antibiotic resistance;
8. Allergens;
The consideration of such cases should include an estimation of the level of expression required to achieve biological or pharmacological activity.

3) Hazards associated with the recipient/host

1. Susceptibility of the host;
2. Pathogenicity of the host strain, including virulence, infectivity and toxin production;
3. Modification of the host range;
4. Recipient immune status;
5. Consequences of exposure;

4) Leakage of engineering bacterial

Many modifications do not involve genes whose products are inherently harmful, but adverse effects may arise as the result of alteration of existing non-pathogenic or pathogenic traits. Modification of normal genes may alter pathogenicity. In an attempt to identify these potential hazards, the following points may be considered (the list is not exhaustive):
1. Is there an increase in infectivity or pathogenicity?
2. Could any disabling mutation within the recipient be overcome as a result of the insertion of the foreign gene?
3. Does the foreign gene encode a pathogenicity determinant from another organism?
4. If the foreign DNA does include a pathogenicity determinant, is it foreseeable that this gene could contribute to the pathogenicity of the GMO?
5. Is treatment available?
6. Will the susceptibility of the GMO to antibiotics or other forms of therapy be affected as a consequence of the genetic modification (Staff & Organization, 2004) (Staff & Organization, 2004) Staff, W. H. O., & Organization, W. H. (2004). Laboratory biosafety manual: World Health Organization. ?

5) Harm of High Frequency Ultrasound

It is reported that the use of cavitational means to create transient membrane pores on living cells (i.e. sonoporation) may potentially induce a broad range of downstream bio-effects that disrupt the functioning of various organelles. (Zhong et al., 2013) (Zhong et al., 2013) Zhong, W., Chen, X., Jiang, P., Wan, J. M., Qin, P., & Yu, A. C. (2013). Induction of endoplasmic reticulum stress by sonoporation: linkage to mitochondria-mediated apoptosis initiation. Ultrasound Med Biol, 39(12), 2382-2392. doi:10.1016/j.ultrasmedbio.2013.08.005

What’s ultrasound-induced membrane permeation phenomenon has been found to be physically instigated through an acoustic cavitation mechanism that involves interactions between ultrasound and gaseous nucleation sites. Even if cell viability can be maintained, sonoporation may still disrupt the normal course of cell cycle progression and cell proliferation
Hence, the effect of high frequency ultrasound should be considered. Ultrasound may upset ER homeostasis, and this may ulti-mately result in initiation of apoptosis

6) Effects of alien bacteria on intestinal environment

The human gut contains more than 100 trillion bacteria, which help the body digest food, produce vitamins protect against disease-provoking bacteria in food, and stimulate the immune system. All these bacteria are separated from the rest of the body by the intestinal wall, which functions as a selective barrier aimed at allowing only useful substances to pass and be absorbed in the body. But when our engineering bacteria break into the intestinal environment, what would happen?
Major functions of the gut microflora include metabolic activities that result in salvage of energy and absorbable nutrients, important trophic effects on intestinal epithelia and on immune structure and function, and protection of the colonised host against invasion by alien microbes. Gut flora might also be an essential factor in certain pathological disorders, including multisystem organ failure, colon cancer, and inflammatory bowel diseases. Nevertheless, bacteria are also useful in promotion of human health (Guarner & Malagelada, 2003) (Guarner & Malagelada, 2003) Guarner, F., & Malagelada, J.-R. (2003). Gut flora in health and disease. The Lancet, 361(9356), 512-519. doi:10.1016/s0140-6736(03)12489-0 .

Fig 1. The particle standard curve obtained form the 2nd calibration experiment.

Reference

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