A novel method for genetic transformation of C. albicans using modified-hydroxyapatite nanoparticles as a plasmid DNA vehicle

Abstract

A new method for delivery of plasmid DNA into Candida albicans using arginine–glucose–PEG functionalized hydroxyapatite nanoparticles as the vehicle which delivers pDNA with high transformation efficiency.

A nano-biomimetic transformation system enables in planta expression of a reporter gene in mature plants and seeds

“…The efficiency of pDNA conjugating to R-nHA was assessed following the procedure of Deshmukh et al 32 Briefly, aqueous 1 mg ml −1 suspensions of R-nHA were prepared by sonicating for 10 min on ice. An aliquot 500 ng of pDNA was mixed with R-nHA suspensions at different w : w ratios (1 : 10, 1 : 30, 1 : 50, 1 : 70, 1 : 100, 1 : 200).…”

“… 25,26 While CaP nanoparticles describe a general type of particle with various Ca/P ratios and acidities, hydroxyapatite (HA) is the most natural and common form of CaP and is similar to the type of CaP found in mammalian bones and teeth. 27 The use of nanoHAs (nHAs) as novel gene carriers has so far been demonstrated in animal cells, 28–30 bacterial cells, 16,31 and yeast cells, 32 but their effectiveness for gene delivery in plants has yet to be shown. More recently, biocompatible nHAs have been successfully used in biomedical systems to deliver diverse molecular cargo 30,33 and in agricultural systems as synthetic fertilizer.…”

Magnesium aminoclays as plasmid delivery agents for non-competent Escherichia coli JM109 transformation

“…The employment of cationic cell penetrating peptides (Toyohara et al, 2019) holds great potential for the delivery of large-sized plasmids, however this approach is not directly comparable to the approach presented in this work since a different strain was transformed (E. coli DH5α), but especially because of the extremely large size plasmid that was used (pMSR227, 205 kb), resulting in a very low transformation efficiency (~ 8 CFU/μg DNA) (Islam et al, 2019). Another approach using arginine-glucose functionalized hydroxyapatite nanoparticles and a 10 kb plasmid has achieved very high transformation efficiencies on Escherichia coli DH5α (10 9 CFU/μg of DNA), Staphylococcus aureus MTCC 737 (10 7 CFU/μg of DNA) (Deshmukh et al, 2019a) and Candida albicans 183 (10 6 CFU/μg of DNA) (Deshmukh et al, 2019b) which is very promising even though a direct comparison with the results reported herein cannot be made without taking into account the fact that different hosts and plasmids were employed. Despite the promising efficiency results, these functionalized nanoparticles are more expensive than aminoclays to synthesize, requiring also a much more complicated and laborious process than Mg aminoclays and a more accident susceptible manipulation of dangerous reagents.…”

“…Despite reports of other methods using DNA delivery agents with similar or even superior efficiency values, the novelty and benefits of a transformation approach are also measured by the cost of the starting materials, the difficulties and safety of production of the delivery agent, health and environmental implications that follow a prolonged or extensive use of all the materials involved and, finally, practical advantages for the end user such as retail price, time gain, level of skills required and fulfillment of several safety and hazardous requirements in comparison to competing methods. The production of Mg aminoclays is more cost-effective, simpler and requires a lower level of safety measures than the production of arginine-glucose functionalized hydroxyapatite nanoparticles (Deshmukh et al, 2019a(Deshmukh et al, , 2019b. As for the end user, since the procedure presented in this work is already optimized and very simple, basically just requiring 1 min mixing for complex formation and 1 min mixing of the DNA-Mg aminoclay complexes with cells followed by spreading into Petri dishes (which avoids any prolonged intermediate steps such as incubation for complexation), it is more userfriendly in terms of time and skills required than other competing methods.…”

“…Therefore, efficient emergent alternative transformation methods and their optimization resulting in routine laboratory protocols are crucial to unravel the potential of recombinant DNA technology which stimulates the development of the field of biotechnology. Novel delivery agents have recently been developed such as a cationic fusion peptide that penetrates Escherichia coli enabling the expression of a large-sized plasmid (pMSR227, 205 kb) (Islam et al, 2019) and arginine-glucose functionalized hydroxyapatite nanoparticles, that can be used for transformation of at least one Gram-negative and one Grampositive bacterial species (Deshmukh et al, 2019a) and one species of yeast (Deshmukh et al, 2019b) with high transformation efficiencies.…”