Preparation of Competent Cells of M. bovis BCG v1

This protocol is a standardized protocol obtained from the Polytechnic University of Hong Kong and is in current use in SC 193 Laboratory under Prof. Au Wing Ngor for M. bovis BCG experimentation. The cells produced thereof are electrocompetent.

Transformation v1

This protocol is used to transform plasmid DNA into competent cells by chemical method or electroporation.

Targeting cAMP-pathway in Regeneration-Competent Cells of Nervous Tissue: Potential to Create a Novel Drug for Treatment of Ethanol-Induced Neurodegeneration

Background: Existing neuroprotective drugs are not effective enough to treat alcoholic encephalopathy. This makes the development of novel pharmacological approaches to treating patients with ethanol-induced neurodegeneration(EIN) relevant. Therefore, the search for new targets among intracellular signaling molecules of regeneration-competent cells of nervous tissue is promising. Objective: This study aims to explore the involvement of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in the realization of the functions of nervous tissue progenitors and glial cells in EIN. Methods: Experiments were conducted on mice of C57B1/6. EIN was modeled in vitro and in vivo. The effects of the adenylate cyclase (AC) and PKA inhibitors on the colony-forming capacity of neural stem cells (NSC) and neuronal-committed progenitors (NCP), their proliferative activity, and intensity of specialization were investigated. The secretion of neurotrophins by astrocytes, oligodendrocytes, and microglial cells was also evaluated. Individual fractions of cells were obtained using the immunomagnetic separation method. Results: The cAMP/PKA signaling is shown to stimulate the proliferation of the NSC and inhibit the mitotic activity of the NCP under the conditions of their optimal vital activity. cAMP reduces the specialization intensity of both types of progenitors. EIN leads to the inversion of the role of the cAMP/PKA-pathway in the regulation of NSC functions. cAMP-pathway has varying influences on the secretion of neurotrophic growth factors by glial cells depending on their living conditions. AC blockage stimulates the realization of the NSC and NCP growth potential and production of neurotrophins by astrocytes and microglial cells in EIN. Conclusion: These findings show the potential for the use of AC inhibitors as novel effective drugs for the therapy of alcoholic encephalopathy.

Metabolic engineering of Bacillus subtilis with an Endopolygalacturonase gene Isolated from Pectobacterium carotovorum; a Plant Pathogenic bacterial strain.

Pectinolytic enzymes [pectinases] produced by microbes are highly important for their biotechnological use in processing of vegetables and fruits beverages and use in pulp and paper industry. A pectinases, namely endo-polygalacturonase [endo-PGase], encoding gene isolated from Pectobacterium carotovorum, a plant pathogenic strain of bacteria was successfully cloned into a secretion vector pHT43 having σ?-dependent promoter P grac . For enhanced expression analysis, competent cells of Bacillus subtilis (WB800N) were prepared at stationary phase using high salt medium. The recombinant B. subtilis competent cells, harboring the engineered pHT43 with the endo-PGase gene were cultured in 2X-yeast extract tryptone medium. The recombinant endo-PGase enzyme was secreted directly into the medium after 72 hours of the first IPTG induction. The recombinant endo-PGase was screened for its activity at various temperatures and pH ranges. Optimal activity was found at pH 5.0 and a temperature of 40°C with a stability ranging from pH 5.0-9.0. For detection of metal ion effect, recombinant enzyme was incubated with 1mM concentration of; Ca ++ , Mg ++ , Zn ++ , EDTA, K ++ for 45 minutes. Resultantly, Ca ++ , EDTA and Zn ++ strongly inhibited the enzyme activity. The chromatographic analysis of enzymatic hydrolysate of polygalacturonic acid [PGA] and pectin substrates using HPLC and TLC revealed that tri and tetra-galacturonates were the end products of hydrolysis. The study led to the conclusion that endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis and assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safe for commercial enzyme production as compared to yeast and fungi to escape endotoxins.

Analysis of the efficiency factors of electrotransformation of Bacillus subtilis to inactivate the aroK gene by the method of homologous recombination

A hyper-osmotic electrotransformation method was developed for strain Bacillus subtilis. Sorbitol and mannitol are included in the hyper-osmotic electroporation medium and recovery medium. In this study, the hyper-osmotic electroporation method was optimised to increase the transformation efficiency of B. subtilis strain 5434 (non-transformable by chemical methods) by 430 fold, with a maximum value of 8.6 ⋅ 105 CFU/µg of integrative plasmid DNA. With the electroporation setted 25 µF, 23 kV/cm, 200 Ω, the method was optimised as follows: a) the OD600 value of the bacterial culture solution was increased to about 1.2, which significantly enhanced survival of bacteria and quantity of viable B.subtilis strain 5434 cells after electroporation; b) the elution frequency of washing solution (hyper-osmotic electroporation medium) for complement cells was increased from 3 to 5 times, resulted in significantly reducing the conductivity of the hyper-osmotic electoporation medium with competent cells (electrocompetent cultue), and effectively extending the pulse time under the same electric field strength; c) quantity of integrative plasmid DNA added to hyper-osmotic electrocompetent culture was optimised. These results indicate that increasing the number of viable B. subtilis strain 5434 cells and reducing the number of metal ions in the electroporation solution mix (integrative plasmid DNA, competent cells of B. subtilis strain 5434, electroporation medium) are useful approach to improve transfomation efficiency of B. subtilis strain 5434. Concentration of shikimic acid in the fermentation medium was quantified by high performance liquid chromatography. Quantification of shikimic acid revealed that B. subtilis strain 5434p4SA produced 403.98 ± 9.1 µg/mL of shikimic acid.

Production of SARS-CoV-2 Virus-Like Particles in Insect Cells

Coronavirus disease (COVID-19) causes a serious threat to human health. Virus-like particles (VLPs) constitute a promising platform in SARS-CoV-2 vaccine development. In this study, the E, M, and S genes were cloned into multiple cloning sites of a new triple expression plasmid with one p10 promoter, two pPH promoters, and three multiple cloning sites. The plasmid was transformed into DH10 BacTMEscherichia coli competent cells to obtain recombinant bacmid. Then the recombinant bacmid was transfected in ExpiSf9TM insect cells to generate recombinant baculovirus. After ExpiSf9TM cells infection with the recombinant baculovirus, the E, M, and S proteins were expressed in insect cells. Finally, SARS-CoV-2 VLPs were self-assembled in insect cells after infection. The morphology and the size of SARS-CoV-2 VLPs are similar to the native virions.

Different simple protocols for separating different types of coexisted plasmids in the same cell

The existence of mixed plasmids in the same cell is tricky and there is a need for separating them from each other. However, isolating two existed plasmids might be difficult, particularly if they are same in their sizes, with same antibiotic marker, or different only in one or more mutants without different restriction cut. Two different plasmids in the same cells is a natural phenomenon as well as a normal practice in molecular biology experiments. For example during random mutagenesis experiment for a single gene (existed naturally in an operon) using a mutator strain like E. coli XL1 Red, the single mutated gene is then complemented with the other essential genes for producing certain products. Another example, during site directed mutagenesis experiment using double antibiotics selection method, in many cases, the original plasmid is existed side by side with the one carry the new mutant. There are many examples where plasmids coexisted with each other either naturally or under experimental conditions. The problem is how one could separate those plasmids particularly when they are similar in their molecular weight and have the same marker. This study introduces two main strategies; the first is based on increasing or decreasing the competent cells transformation efficacy. Where, in general harvesting competent cells either E. coli or other bacterial strains in the first 2-3 hours (or less) of their cultivation and using the enhanced protocol for competent cells preparation will improve the transformation processes. Letting cells to be more ages will reduce the transformation processes. Using four 2-3 hour grown competent Azotobacter sp enable plasmid transformation. The second strategy for separating the coexisted plasmid is based on using diluted plasmids. The antibiotic screening method is based on blind selection where growing on plat containing the first antibiotic and non growing in the second antibiotic means that the tow existed plasmids are separated. In case of existing of plasmids with the same size and the same antibiotic marker for example during the site-directed-mutagenesis protocol (mutants did not have different restriction enzymes cut), the plasmid is diluted and transformed in recombinant E. coli and each clone was cultivated alone and the mutated region is sequenced. The presence of a single base pair in the site of the mutant means presence of a single plasmids and vice versa. As a conclusion same plasmids with point mutation are usually coexisted. In some cases the coexisted plasmids are with similar antibiotic marker, no different restriction enzyme cut sites are existed, no white and blue selection or any other phenotype for selection. In such cases and similar ones diluting plasmid and transforming them in conditions enable single plasmid per cell must be controlled by the sequencer. The protocols included in this study are summarized from the experiences with random and site directed mutagenesis experiments where plasmid with a single mutant is coexisted with the wild mother plasmid or with the other coexisted different mutants.