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Artificial Transformation

Artificial transformation methodologies for improving the efficiency of plasmid DNA transformation.

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Artificial Transformation

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  1. Artificial transformation methodologies for improving the efficiency of plasmid DNA transformation. BY:SAEED S. ALSMANI M.Sc. Biotechnology B.Sc. Microbiology

  2. PRESENTATION FLOW • INTRODUCTION •    CHEMICAL TRANSFORMATION •    ELECTROPORATION •    PHYSICAL TRANSFORMATION •    OTHER TRANSFORMATION METHODS •    COMBINED TRANSFORMATION METHODS •    CONCLUSION

  3. INTRODUCTIONBacteria can naturally transfer genetic material in three ways:CONJUGATIONTRANSDUCTION TRANSFORMATION

  4. Taking naked DNA into the cell • When a bacterial cell dies, genetic material is released into the environment. This genetic material can then be taken up by a living bacterial cell. • Receiving of exogenous DNA substances by bacteria through the cell membrane is known as transformation. • It is essential for the genetic manipulation of bacteria and is therefore important in biotechnological and biological research.

  5. The ability of a cell to taking extracellular DNA ("naked DNA") from the medium is called "competence". However, not all bacteria can take exogenous DNA from their environment. • Since the efficiency of natural transformation is very low, various artificial transformation methods have been developed for simple and effective bacterial transformation.

  6. Chemical transformation is based on chemical processes that weaken cell membranes using cations (eg. Ca 2+) and low temperature. (Mandel and Higa 1970). • In the 1900’s, it was first discovered that some strains of Escherichia coli (E. coli) could receive large amounts of DNA in the presence of calcium ions. After this discovery, the use of the method of chemical transformation based on CaCl2 became popular in laboratories. (Dagert and Ehrlich 1979; Mandel and Higa 1970)

  7. The use of monovalent ions (Li +, Na +, K +), bivalent ions (Ba2 +, Ca2 +, Mn2 +, Sr2 +) and trivalent ions (Al3 +) for transformation have been analyzed in various bacterial species, and has been found that cells using bivalent ions have transformation ability.(Liu et al. 2014).

  8. The cations bind to phosphate groups (in DNA) and other negatively charged groups on the bacterial cell membrane to allow plasmid DNA (pDNA) to adhere to the cell membrane.Then a sudden increase in temperature creates pores in the plasma membrane of bacteria and allows plasmid DNA to enter the bacterial cell.

  9. The transformation efficiency depends on several factors including cation type, cation concentration, thermal shock and incubation time. • E. coli was best modified under the following conditions: • cation concentration (40-60 mM MnCl2), • thermal shock (40–50 ° C), • incubation time (40–60 s).

  10. MODIFICATIONS • Transformation efficiency can be further enhanced by incubating the cells in a cold CaCl2 solution for a longer period of time. (Dagert and Ehrlich 1979). • A more efficient transformation of gram-positive bacteria was achieved by treating gram-positive bacteria with the lysozyme enzyme, which removes the thick cell wall of gram-positive bacteria.

  11. ANIMATION • https://youtu.be/w8szG7QOC5k

  12. Electroporation involves the use of high-voltage electrical impulses to destabilize the cell membrane, thereby triggering the formation of temporary pores. • This method is widely used in laboratories because its transformation efficiency is higher than other methods.

  13. In the cell membrane, the phospholipids are aligned in two layers, where the heads are collected in the same area, and the hydrophobic tails are gathered on another side. when we apply a small amount of electricity, it realigns all these phospholipids. (as in figure)

  14. The main advantage of electroporation can be applied for all cell types. Moreover, it can transform many cells in a short time. • the main disadvantage is the possibility of cell death from high-voltage impulses.

  15. MODIFICATIONS • Recently, There is a new improved device that controls the electric fields by inducing high electric fields with small voltage.

  16. ANIMATION • https://youtu.be/NxGpLYdi1uM

  17. Physical transformation occurs by using a colloidal solution consisting of Nano-sized acicular substance and bacterial cells, then is stimulated by friction between the hydrogel and the interfacing material.

  18. The Nano sized acicular materials accompanies pDNA to form a complex. • This complex grows and due to the driving force caused by friction penetrates bacteria cells. This effect is known as the “Yoshida effect” in honor of its discovery. (Yoshida 2007). • Nanoparticles such as chitosan, GNPs and CNTs were used to provide pDNA. (Kumari et al. 2017; Maoet al. 2001; Rojas-Chapana et al. 2005)

  19. Ultrasound transformation • Ultrasound method for gene transfer has recently been used in eukaryotes. the principle is based on the cavitation effect that creates reversible porosity in the cell membrane. • The bioactivity of this technique is similar to electroporation and is superior in some cases. • Prolonged exposure of ultrasound to low frequency (<MHz) has been shown to cause cellular death.

  20. The micro shock wave device is a new device for transformation, which can instantly increase the pressure and temperature of the medium for transforming the cell. This method has been used for the transformation of Pseudomonas aeruginosa and Salmonella typhimurium. • Freezing-thawing bacterial transformation method. It has the ability to freeze and thaw cells twice using liquid nitrogen. It was used for E. coli. • (Tripp et al. 2013)

  21. Combined methods have been developed to increase the applicability of transformation technologies to various bacterial species. • (Electro-Chemical) Method • In 1999 the combined (Electro-Chemical) method was reported, in which the thermal shock is applied after electroporation. • The transformation efficiency of Gram positive bacteria (Corynebacterium glutamicum) was increased 100-fold after electroporation with low growth temperature and thermal shock.

  22. (Physico-Chemical) Method • This method can be applied to various types of bacteria, including both Gram positive and Gram negative bacteria. • Chemical cations make the cell membrane permeable and nanomaterials form Nano channels from the cell membrane. • Physicochemical transformation was further optimized using 5 different chemical compounds (RbCl, DMSO, MgCl2, LiAc and CsCl) and 4 different nanomaterials (sepiolite, gold (III) chloride, multi-walled CNT and chitosan). • (Ren et al. 2018).

  23. DNA transformation is a basic process for genetic manipulation and bacterial engineering. • Various artificial transformation methods based on chemical compounds, nanoparticles, electric shock and other systems have been developed to increase the effectiveness of E. coli and industrially attractive bacteria species, since artificial transformation methods will not always provide good results for newly defined bacterial species or provide a lower transformation efficiency. • Therefore, there is still a demand for simple and universal transformation methods that can be applied to various types of bacteria.

  24. REFERENCES Berthier F, ZagorecM, ChampomierVergesM, Ehrlich SD,MorelDeville F (1996) Efficient transformation of Lactobacillus sake by electroporation. Microbiol-Uk 142:1273–1279. https://doi.org/10.1099/ 13500872-142-5-1273 Bozkir A, Saka OM (2004) Chitosan-DNA nanoparticles: effect on DNA integrity, bacterial transformation and transfection efficiency. J Drug Ta rge t 1 2 ( 5 ) : 2 8 1 – 2 8 8 . h t t p s : / / d o i . o rg/ 1 0 . 1 0 8 0 / 10611860410001714162 Brito LF, Irla M, Walter T, Wendisch VF (2017) Magnesium aminoclaybased transformation of Paenibacillus riograndensis and Paenibacillus polymyxa and development of tools for gene expression. Appl Microbiol Biotechnol 101(2):735–747. https://doi.org/ 10.1007/s00253-016-7999-1 Campos-Guillen J, Fernandez F, Pastrana X, Loske AM (2012) Relationship between plasmid size and shock wave-mediated bacterial transformation. Ultrasound Med Biol 38(6):1078–1084. https://doi.org/10.1016/j.ultrasmedbio.2012.02.018 Chan WT, Verma CS, Lane DP, Gan SK (2013) A comparison and optimization of methods and factors affecting the transformation of Escherichia coli. Biosci Rep 33(6). https://doi.org/10.1042/ BSR20130098 Chandrasekaran G, Han HK, Kim GJ, Shin HJ (2011) Antimicrobial activity of delaminated aminopropyl functionalized magnesium phyllosilicates. Appl Clay Sci 53(4):729–736. https://doi.org/10. 1016/j.clay.2011.07.001 Chang S, Cohen SN (1979) High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet 168(1):111– 115. https://doi.org/10.1007/bf00267940 Choi HA, Lee YC, Lee JY, Shin HJ, Han HK, Kim GJ (2013) A simple bacterial transformation method using magnesium- and calciumaminoclays. J Microbiol Methods 95(2):97–101. https://doi.org/10. 1016/j.mimet.2013.07.018 Chuanchuen R, Narasaki CT, Schweizer HP (2002) Benchtop and microcentrifuge preparation of Pseudomonas aeruginosa competent cells. Biotechniques 33(4):760, 762-3. https://doi.org/10.2144/ 02334bm08 Chung CT, Niemela SL, Miller RH (1989) One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A 86(7):2172–2175. https://doi.org/10.1073/pnas.86.7.2172 Cohen SN, Chang AC, Hsu L (1972) Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by Rfactor DNA. Proc Natl Acad Sci U S A 69(8):2110–2114. https:// doi.org/10.1073/pnas.69.8.2110 Dagert M, Ehrlich SD (1979) Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6(1):23– 28. https://doi.org/10.1016/0378-1119(79)90082-9

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