Efficacy of genetic transformation of E.coli field and reference (ATCC 8739) strains at different concentrations of CaCl 2 for creation of Gene libraries

. The importance of genetic transformation in various fields of biological research, and especially in the process of gene libraries formation, is very important. It has been proven by numerous studies that during the phase of obtaining competent cells, Ca 2+ ions neutralize the electrostatic repulsion of membrane proteins and DNA of the bacterial cell, thereby stimulating the introduction of foreign DNA into the membrane pores. Our research aimed to elucidate the maximum efficiency of transformation in field and ATCC 8739 reference strains of E.coli at 0.05; 0.1 and 0.15 M concentrations of CaCL 2 . Microbiological, molecular-biological modern methods were used in the experimental studies. From the research results, it became evident that the efficiency of genetic transformation increases with the addition of concentration of CaCL 2 and reaches the maximum at 0.1 M. Among the studied strains of E.coli , at 0.05 and 0.1 M concentrations of CaCL 2 , the highest efficiency was recorded in the field strain. At a 0.15 M concentration of CaCL 2 , the transformation efficiency in the field and reference strains is highly variable due to the size of the genomic DNA of the internalizing bacteria and the number of restriction sites.


Introduction
Genetic resources are considered the reachness of every country.Formed as a result of natural evolution and human activity, they are very important in the process of food production and environmental balance [1].
At the current stage of agricultural development, when the task is to create new varieties of crops that meet population and production requirements in a short period of time, or to improve existing ones, conservation and efficient use of genetic resources is considered as a strategic task.
Currently, the most practical method of conservation, inventory, location and acquisition of valuable gene pools is the creation of DNA banks or gene libraries.A gene library is a group of living bacterial colonies built in a vector containing restriction sites of the genomic DNA of a single organism, which are considered a source of desired genes.
In other words, the total DNA segments of valuable species are stored in bacterial cells.It was created for the first time in 1974 by D. Khognes and employees.Practically every bacterial cell is deploying one or more genomic DNA segments that represent a hybrid of one of the many restriction segments of vector or DNA.The bacterial cell, receiving the hybrid DNA, multiplies to produce a clone.A collection of bacterial cells containing all the restriction fragments of the genomic DNA of a particular organism forms a DNA bank [2].
The whole process of creating gene libraries includes a series of operations, where the genetic transformation of bacteria is essentially the most important, because their efficiency is directly proportional to forming a complete DNA bank [3].
The phenomenon of transformation was first discovered in 1928, by F. Griffith [4].It is a change in the hereditary information of a bacterial cell due to the introduction of DNA segments from another cell or another strain into it.In the laboratory, transformation is used to introduce recombinant plasmid DNA into bacterial cells, in which case the plasmids replicate independently of the chromosomal DNA and ensure the expression of the target genes.Transformation provides an opportunity to introduce genetic constructs that, by recombination with chromosomes, can be integrated into it and reproduced together (integrative plasmids) [5][6].
The initial material for many biological studies is the acquisition of cells containing foreign DNA, this phenomenon is considered genetic transformation.It makes it possible to obtain genetically modified organisms, combine or disable the necessary genes, determine the effect of certain proteins and finally create gene libraries or genetic banks [7].
In many cases, due to research purposes, it is necessary to accurately determine the efficiency of transformed cells, which is the ratio of the total number of transformed cells to the amount of DNA used for transformation.The efficiency of transformation is directly related to obtaining competent cells.E.coli cells capable of including foreign DNA into themselves are considered as a competent.One of the most common methods for obtaining competent cells is the incubation of bacteria at low temperature in a liquid medium containing Ca 2+ ions [8][9].
The method is known as cell calcinations.The nature of the method consists in the following: calcium ions bind to the outer membrane of cells and contribute to the adsorption of foreign DNA due to ionic bonds.The maximum level of adsorption is maintained for 2 hours after incubation, after which it gradually decreases, due to which the transformation efficiency drops sharply [8].
Both the size of the vectors, the type of E.coli and the amounts of CaCL 2 used for cell calcination have some influence on the transformation efficiency.In this work, the effect of CaCL 2 with different molarity (0.05, 0.1 and 0.15 M) on the transformation efficiency of reference (ATCC 8739) and field strains of E.coli is presented.The results can be used as a test in the process of genetic transformation and especially the creation of gene libraries in various fields of biology.

Materials and methods
The research was carried out in 2021-2023, in the Biological Research Laboratory of the "Scientific Center of Agro-Biotechnology" branch of the Armenian National Agrarian University /ANAU/.The samples of the studied plants were taken from the ex situ seed collection of the National Genebank of Crops and their Wild Relatives of the Scientific Center.Genomic DNA from crops and wild species was obtained by the SDS method [10].The concentration of extracted DNA was determined using a NanoDrop One (Thermo Scientific, USA) spectrophotometer.
Ampicillin resistance selection of E.coli was carried out in order to obtain colicinogenic plasmids against ampicillin (as a selection marker) [11].
Colonies of E.coli were grown in 100 ml of LB medium with ampicillin (50 μg/ml) and incubated at 37°C for 18 hours.A portion of the overnight culture was transferred to fresh LB medium containing ampicillin, ensuring its 100x dilution.Growth was continued for 16-18 hours until the appropriate optical density (2-3*10 8 cells/ml) was reached.
When a single restriction enzyme is used, very often the cleaved ends of the vector stick together, forming the vector's starting forms without large amounts of internalized DNA.In order to avoid all this, dephosphorylation of plasmid DNA was carried out immediately after restriction according to the following scheme: 5 µl of 10x buffer was added to 50 µl of the reaction mixture (5 µg DNA, 50 µl deionized water, 2 µl alkaline phosphatase).The mixture was incubated at 37 0 C, for 1 hours.DNA purification was carried out by phenolchloroform method, phosphatase was removed by 96% ethanol.
The number of competent cells was determined by the following formula [14]: Where: K -is the number of competent cells, N -total number of colonies per Petri dishes.
Genetic transformation of E.coli cells was carried out by heat shock method [17].
The transformation efficiency was determined by the following formula [14,18]: Where: T -is the transformation efficiency, M -total number of transformed colonies per Petri dishes, A -amount of built-in DNA (µg).
The results of DNA incorporation into transformed cells were determined by the restriction enzyme method [10].

Results and Discussion
In order to investigate the transformation efficiency of different strains of E.coli and different concentrations of calcium chloride, field and reference strains of E.coli were treated with different molar solutions of CaCL 2 to increase the competence of strains.The maximum transformation efficiency was observed in the field strain of E.coli when treated with 0.1 M solution of CaCL 2 (Table 1).
The lowest concentration efficiency was recorded when the reference strain of E.coli was calcinated with a 0.05 M solution of CaCL 2 .In the case of 0.15 M calcium chloride solution, high transformation efficiencies were reported for both field and reference strains, with a highly defined pattern depending on the genome size of the transformed plants and the number of genomic DNA restriction fragments (Table 1, Figure 1).Therefore, we hypothesize that the efficiency of transformation depends not only on the type of E.coli, the concentration of Ca 2+ ions required to neutralize the negative charges of their cells and plasmid (vector), but also on the number of transformed clones.
Cells were not ampicillin resistant prior to transformation.To establish that Ca 2+ ions are very important in the process of obtaining competent cells, non-calcified cells were transformed and grown in nutrient medium containing ampicillin.The transformation efficiency was equal to 0 in the case of both strains of E.coli.
Studies have shown that the presence of Ca 2+ ions is essential for obtaining competent cells.From the analysis of the materials obtained as a result of our research, it becomes clear that during transformation, the electrostatic repulsion between the negative charges of the bacterial outer membrane and the DNA prevents the proximal association, which in his turn reduces the transformation efficiency.The presence of positively charged Ca 2+ ions promotes transformation activity by neutralizing the negative charge associated with DNA and the cell membrane and thus reducing electrostatic repulsion.The aim of our studies was to determine the effect of different strains of E.coli and different molarity of CaCL 2 on transformation efficiency.From the results of the studies, it is clear that at a low concentration of CaCL 2 (0.05 M), the highest transformation efficiency was recorded in the field strain of E.coli (4x10 6 -28x10 6 ).Under the conditions of the same concentration of calcium chloride, the efficiency of transformation of E.coli reference strain in all versions ranged from 3x10 6 -20x10 6 .It is noteworthy that, when comparing the averaged data, the field strain of E.coli at 0.05 M CaCL 2 exceeds the reference strain by 28.1% in transformation efficiency.Upon calcination with a 0.1 M solution of calcium chloride, the transformation efficiency is dramatically increased in both studied strains.For the field strain of E.coli, it ranges from 2x10 8 to 3.6x10 8 (average: 2.6x10 8 ).In the 0.1 M version of CaCL 2 , the field strain outperformed the reference strain by 18.75% in transformation efficiency.In fact, as the CaCL 2 concentration was doubled, the transformation efficiency increased dramatically.
Scientific sources prove that high concentrations of CaCL 2 create an environment of high osmotic stress, leading to upregulation of non-specific outer membrane proteins.Outer membrane protein C (OmpC) is a variant of a nonspecific solute, porin, whose treatment with CaCL 2 facilitates the uptake of foreign DNA.From this reasoning, it can be concluded that the reference strain of the E.coli is more resistant to osmotic stress compared to the field strain; therefore less OmpC is produced to mitigate it, which may be the reason for the relatively low transformation efficiency.However, this pattern is not maintained at 0.15 M concentration of CaCL 2 [19][20].
In the case of genetic transformation of restriction segments of plant species with gene sizes of 2.5x10 6 -17x10 6 , the efficiency of transformation is high in the reference strain, it ranges from 32x10 6 -68x10 6 (average: 50x10 6 ).In the field strain, it ranges from 24x10 6 to 48x10 6 (mean: 36x10 6 ), falling by 28%.
The efficiency of the vector insertion process containing the restriction regions of plant species with genome sizes of 157x10 3 -989x10 3 is high in the field strain of E.coli, ranging from 35x10 6 -84x10 6 (average: 59.5x10 6 ).In these variants, the transformation efficiency falls short of the reference strain, ranging from 27x10 6 to 60x10 6 (average 43.5x10 6 ), falling by 27% (Table 1, Figure 1).
Regardless of the fact that the efficiency of transformation due to the size of the genome of plant species, and therefore also the number of restriction segments, is highly variable in the reference and field strains of the E.coli, it should be noted that, in general, the efficiency of transformation at a concentration of 0.15 M of CaCL 2 is very low compared with the 0.1 M. In this assay scheme, transformation efficiency in both E.coli strains increases dramatically from 0.05 M to 0.1 M CaCL 2 at the expense of increased OmpC protein transcription, after which it decreases markedly.At a 0.15 M concentration of calcium chloride, the decrease in transformation efficiency is a consequence of high osmotic stress, which forces cells to limit the amount of uptake of foreign DNA, since the latter is not considered an essential component for cell survival.

Conclusion
Summarizing the results of the experiment, it can be concluded that the efficiency of genetic transformation in reference and field strains of E.coli is the highest at 0.1 M concentration of CaCL 2 .Incidentally, the field strain has a significant advantage in transformation efficiency at both 0.05 and 0.1 M CaCL 2 concentrations.Regarding the conflicting results obtained at the 0.15 M concentration, which we believe is due to the amount of genomic DNA and the number of restriction sites of the introduced plant species, further studies are needed, and therefore we refrain from drawing conclusions.

Fig. 1 .
Fig. 1.Dependence of transformation efficiency on genome size for different strains of E.coli at 0.15 M concentration of CaCL 2 .

Table 1 .
Transformation efficiency due to different strains of E.coli and different concentrations of CaCL 2 .