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  January 31, 2023
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From a sample CHAPTER for the publication: "Topics in Biotechnology and Animal Nutrition for Feeding and Production," - Vol III-in the set, (c) D. A. Flores. Skye Blue Publications. Port Coquitlam, B. C. Canada V3B 1G3. 

Chapter 13. Biosafety in Genetic Manipulation of Microbes and Plants.

The Problem and the Potential.

De-risking the event of genetic manipulation either via gene recombination or cloning or by the newer method of gene editing (e. g. CRISPR/Cas9) is considered generating genetically modified organisms (GMO) and poses a potentially biosafety breach due to contamination and disturbance of ecological balance in the environment or niche of the organisms in question. In this case we address e. g. in our repertoire regards microbial (e. g. bacterial, yeast and fungal), plant (e. g. marine seagrasses) and animalia (e. g. dairy cows) that require genetical manipulation and modification to bring about process improvement, productivity in growth in plants or with pharma applications or in terms of what they produce in animal products.

Either biosafety is required in: a given animal physiological compartment, biosafe plant growing facility or in a physical sense or via providing an alternative and what is considered, a non-GMO generating approach to a backgrounded genetic manipulation, such as technique of protoplast fusion. 

We will now discuss the following possibilities as illustrated with each example following.

GMO Probiotics and the Porin Model in Rumen Cow Stomach Biocontainment.

By the porin model used to contain GMO probiotics in the rumen stomach of cows we define the following stipulations: proteins used as "plugs" on what would be pores causing influx/efflux of electrolytes, causing the cell to also undergo osmotic shock, bursting it eventually, which will be produced to transbound the microbial cell membrane and its pore protein subunits, e. g. in a hexa- or pentapeptide complex) designed using nanotechnology of specific diameters (in the magnitude range of Angstroms) that closely fits a plugged hole model. A pharma approach will be used in media that causes sporulation and porin plug formation with the spores fed as pellets or tablets with chewing the porin biopharmaceutical. Additionally another biosafety measure can be used in the way of immunocontrol via vaccination of associated animals with quarantine and of animal handlers exclusive to the flock or herd in direct contact with the quarantined facility which it is ecpected will result in further biosafety control of probiotic-fed animals.

GMO Marine Grasses and Secondary Alkaloids Against Pollination.

Colchicine, podophyllotoxin and vinblastine are plant secondary alkaloids that bind to cellular microtubular protein structures and act as agents that stop the birefringence of mitotic spindle fibres and division in the pollen mother cells, specifically in reference to Lilium. The next step is to produce a biocontainment system based on this secondary alkaloid mechanism bioengineered in preformulated solution from pre-filtered seawater-based media, to bind the mother cell microfibrils that are essential for cell meiosis developing into grains of pollen. In effect, this is expected to result in blocked pollination in sub-hydroponic culture in flowering marine grass species and thus possible escape into the environment as a general but biosafe precaution. Another approach that is "looming in the horizon" is that of maternal segregation in terms of gene recombination, for e. g., producing pharma like insulin that does not cross-pollinate the recombinant DNA or gene from the source plant host.

Non-GMO Protoplast Fusion of Marine Grasses.

Protoplast fusion has been used as a breakthrough method in the past and is considered recombination but of the non-GMO kind wherein the DNA of two mother cells stripped of their cell wall covering fuses their plasma membrane and proceed to exchange or recombine their DNA bringing about a new variety or genus of either microbial or plant cells, in this case seagrasses from marine environments through callus development in vitro and bringing about and by examination their fusants from either parent using X-ray mutation to bring about, for e. g., a genetic change in growth rate, which we will dub here an XL Grow variety, for e. g., as in auxin production via transcription factor (TF) mutation.  There will be a requisite need to stabilize the genetic background of the new organism through several backcrosses with the parental strain until the mutated protoplast fusant is deemed stable as a genetic resource.

Non-GMO Gene Silencing with PNA-Carrier Technology in Microbial Feeds Processing and Rumen Probiotics.

The application of peptide nucleic acid (PNA) with carrier (e. g. vitamin B12 in bacteria) upon "direct application" to bring about gene silencing to problems in feeds processing (e. g. ensilage and ureolytic ensilage of residue-type byproduct feeds) and probiotics in the rumen, is still in its infancy. We will call the system a particulate carrier system (PCS) here for either bacteria, yeast or fungal culture using a mitogenic microbial cell model for microbial proliferation and deproliferation using gene silencing technology. For approaches: ensilage will entail boosting fibrolysis and lactic acid acidification from sugars produced, decreasing the buffering capacity (Bc), controlling clostridial, butyrate-type fermentation, curtailing yeast aerobic deterioration, boosting ammoniation in ureolytic residual silages in the tropics; and in the rumen will entail boosting water-soluble carbohydrates (WSC) with yeast, controlling bacterial proteolytic species and deamination, boosting essential amino acid synthesis in yeast and boosting "surrogate" protein in host spp. in the rumen.

A Novel Non-GMO Alternative Approach to Boost Milk Food Proteins (MFP) in Cows.

The same mechanisms described above is proposed with "directly applied" PNA-B12 gene silencing technology through sub-lingual application or introduction to boost protein production in the udders of dairy cows for boosted MFP.  There is a need to further establish the viability of the technique in terms of also entering effectively the transmembrane barrier in the dairy udder or milk glands of cows.


All approaches described as discussed are at only an initial stage of genetic manipulation towards productive ends. Nevertheless, it does not preclude measures as presented above to consider the biosafe "containment" or alternative non-GMO method or approach to ensuring to de-risking the situation in future when carrying out such genetic transformation for various microbial processes in feeds and also in animalia.


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Last update of this entry: October 23, 2021

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