High yield minicell and protein producing bacterial strains
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Solution Overview
Problem
There is a need for delivery vectors that can efficiently target and deliver biological agents to cells, including animal, plant, and insect cells, with improved efficacy, and methods to modulate biological systems effectively.
Innovation Solution
The development of engineered bacterial cells, specifically E. coli strains with genetic mutations, to produce high yields of minicells that are substantially free of viable parental cells, incorporating insecticidal proteins such as Pir and Cry, and formulations with Bacillus thuringiensis-derived active ingredients, which are administered in synergistic ratios to enhance insecticidal activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional bacterial cells are used to produce minicells, then the production process is simple, but the yield is low and viable parental cells contaminate the minicell population
Solution Approach 1:
The patent divides the bacterial cell into two functional parts: minicells containing the desired biological agents and viable parental cells. By inducing asymmetric cell division through specific genetic modifications (mutating cell division topological specificity factors like minC, minD, minE, or ftsZ), the bacterial population is segmented into numerous small minicells (1-10 µm) and fewer larger parental cells, enabling high yield production while maintaining purity through size-based separation
Solution Approach 2:
The patent extracts the minicells from the parental bacterial cells through a separation process. The minicells, being smaller and lacking viable parental cell machinery, can be selectively removed from the culture mixture using filtration, centrifugation, or density gradient techniques, resulting in a pure minicell population free of viable contaminants
Solution Approach 3:
The patent changes the physical and genetic parameters of the bacterial cells to achieve high minicell yield. Specific genetic mutations in cell division topological specificity factors alter the division mechanics, while additional mutations in DNA repair genes (mutS, MutH, MutL, SSB, RecA, RecJ, ExoI, ExoVII, ExoX, UvrD, or DNA polymerase III) prevent minicell regeneration, creating a one-time division event that maximizes minicell production before cell death
2Quantity of substance
If high expression cassettes are used in parental cells, then protein expression levels increase, but the complexity of the genetic system increases
Solution Approach 1:
The patent employs universal expression cassettes that can be inserted into the minicell-producing parental cells to express various biological agents. The expression cassette includes a promoter (such as T3, T7, K11, or SP6), a ribosome binding site, and the gene of interest, allowing the same basic structure to produce different proteins (e.g., insecticidal toxins Pir and Cry, or other biological agents) by simply changing the inserted gene, thus achieving high protein expression without proportionally increasing system complexity
3Productivity
If minicells are produced with high yield, then the concentration of viable parental cells decreases, but the separation process becomes more challenging
Solution Approach 1:
The patent exploits the size difference between minicells (1-10 µm) and parental cells (larger) to facilitate separation. The smaller minicells can be selectively removed through size-based separation techniques such as filtration through membranes with appropriate pore sizes, centrifugation at specific speeds, or density gradient centrifugation, making the separation process straightforward despite high minicell concentrations
Data Source
AI summary
The present disclosure relates to mixtures of insecticidal minicells containing the insecticidal protein toxins Pir and Cry, and methods related to producing and using the same. The present disclosure also relates to mixtures of insecticidal minicells containing the insecticidal protein toxin Pir with a Bt-derived active ingredient, and methods related to producing and using the same.