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  June 17, 2024
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Chapter 13. Biosafety in Genetic Manipulation of Microbes and Plants.

by D. A. Flores

The Problem and the Potential.

De-risking the event of genetic manipulation either via gene recombination or cloning or by the new method of gene editing (e. g. CRISPR/Cas9) is considered generating genetically modified (GM) or 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 with growth of plants and with animal livestock or for pharma.

Either biosafety is required for animal physiological compartments, a biosafe plant facility in a physical sense or providing alternative non-GMO approaches to genetic manipulation, for e. g., such as the technique of protoplast fusion. 

We will now discuss with the ff. illustrated e. g. s. possibilities for applied problems in areas of production and feeding: 

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

The porin model with GMO-type probiotics in the rumen stomachs of cows are defined with the following stipulations: proteins used as "plugs" on what would be pores causing influx/efflux of electrolytes, causing the cell to undergo osmotic shock, bursting the cell, which will be produced to transbound the microbial cell membrane and the pore protein subunits, e. g. in hexa- or pentapeptide complexes) with the nanotechnology field of specific magnitude in the range of Angstroms that closely fits the plugged hole cell model.  A pharma approach with plug holes will be used with microbial media causing sporulation and porin plugs when spores are fed in pellet form or tabs with chewing.
Additionally requisite biosafety measures applied to a quarantined facility will use immunocontrol via vaccination of associated animal units together with quarantine animal handlers exclusive of the flock or experimental herd also in the quarantined facility.  It is further stipulated that strict biohazard protocols including biocides in the surrounding zonal perimeter is legally apropos according to legal stipulation as enforced. 
Likely, the probiotic technology is headed from Australia to a country like India or Pakistan based on earlier preliminary research on microbial organisms from the rumen stomach. 

GMO Marine Grasses and Anti-Pollinator Secondary Alkaloids.

We believe this classified technology will remain preliminary in standing only at this time. 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 referenced to Lilium with research in the literature. The next step is to construct the biocontainment system with proto-plant rearing reactors using the secondary plant alkaloid mechanism formulated in solution from pre-filtered seawater or saline media, to bind the mother cell microfibrils that are essential for cell meiosis developing into grain pollen.  This is expected to result in blocked pollination in submerged culture for flowering marine grass species and the escape to the environment as a biosafe measure.
One technology that is "looming in the horizon" is that of producing pharma like the drug insulin that does not cross-pollinate the recombinant DNA or gene from the source host.

Non-GMO Protoplast Fusion of Marine Grasses.

Protoplast fusion has been used as a breakthrough method in the past and is considered a recombination event but of the non-GMO class wherein the DNA of two mother cells stripped of their cell wall fuses their plasma membrane together and proceed to recombine DNA bringing about a new variety or genus of either microbial or plant cells.
In the case of seagrass from marine environments through calli development in vitro and bringing about by examination fusants from either of the parental combination, X-rayed by mutation to bring about genetic change in growth rate, the raison d'etre in seagrass proto-culture, which will be dubbed here the "XL-Grow" varieties, for e. g., with auxin boosting using transcription factor (TF) mutation in transcription factor engineering (TFE).  
There will be a requisite need to stabilize the genetic background of new organisms through backcrossing with the parental strains 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 Pre-treatment (physico, bio and chemo) and Rumen Probiotics.

The application of peptide nucleic acid (PNA) with RNA carrier (for e. g. vitamin B-12 in bacteria) upon direct application to bring about the classic phenomenon or mechanism of gene silencing to problems with feeds (e. g. ensilage and ureolytic ensilage of residual byproducts) and those probiotics in the rumen, still in its infancy. We will call the system the particulate carrier system (PCS) coordinate with a biofilm model here for bacteria, yeast or fungi using the mitogenic microbial cell model for microbial proliferation or deproliferation with gene silencing technology. For various approaches ensilage will entails boosting fibrolysis, related lactic acid acidification from sugars produced, decreasing the buffering capacity (Bc), controlling clostridial, butyrate-type fermentation, curtailing yeast aerobic deterioration and associated DM losses, and boosting ammoniation in ureolytic residual silages.  In the rumen approaches will entail boosted water-soluble carbohydrates (WSC) from yeasts, decreasing bacterial proteolytic species with deamination, boosting metabolic essential amino acid synthesis for yeast and challenging prospect of boosting the "surrogate" protein content of host rumen spp.

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 to boost protein production of the udders for dairy cows or goats boosting milk food products (MFP).  
There is a need to further establish viability in the technique in terms of entering the transmembrane barrier in the dairy udder or milk glands cows as another alternative route.


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 de-risking the situation in future when carrying out such genetic work for various microbial processes in feeds and also for animalia.

(c) D. A. Flores. 2023-2050. Skye Blue Internet. Port Coquitlam. B.C. Canada V3B 1G3.

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