Saccharomyces cerevisiae Ghosts Using the Sponge-Like Re-Reduced Protocol

NaOH, SDS, NaHCO3 were prepared as 10% sterile stock solutions. SDS, NaHCO3 were sterilized using the autoclave. NaOH did not autoclaved while it is sterile by itself at 10% concentration. H2O2 was purchased from local pharmacy as 30% sterile solution. 0.5ml of each (of the above-described compounds) was used to conduct the serial dilution experiment. For each compound 0.5ml was added to the first tube in which, the tube contains 4.5ml of the above described broth cultivation medium. 0.5ml was then transferred from the first test tube to the second one and so on until seven tubes for each compound. The tubes were then mixed gently. For the last tube, 0.5ml was discharged. Each tube was then inoculated with 100μl of the freshly prepared (108CFU/ml) S. c. culture. The tubes then mixed gently and left for 48h. The S. c. growth was observed by increasing the amount of sediment cells in the test tube bottom. Abstract


Introduction
Microbial Ghosts are the microbes after their evacuation from their cytoplasmic content without damaging their 3D structures.Yeast is an old microbe used in traditional biotechnology [1].Yeast has showing increasing interest in various biotechnological applications due to its different properties, which were extended by the genetic engineering and molecular biology tools [2][3][4][5][6][7].Yeast is an eukaryotic microorganism which has some unique properties different from bacterial cells [2,[8][9][10].Being eukaryotic, yeasts enable the correct expression for different types of recombinant proteins [2,11,12].For more information about the different human proteins produced and expressed in the yeasts refer to Service (2003) and the references within [13].Regarding to its big size the yeast cell could be used as drug delivery system [14][15][16][17][18][19].Yeast Ghosts preparation was reported using some conformational isomers of pancreatic [20].This study

S.c.Gs Preparation Using Re-Reduced SL Protocol
One gram of the dried yeast cells was added to 10ml of the MIC of the NaHCO 3 and subjected to gentle shaking for one hr.Then the yeast cells left static to enable the cells to sediment.Sample from the supernatant was taken to determine the amount of the realized DNA and Protein.The supernatant then discharged and the cells washed by distilled water, left for decantation and the supernatant discharged.The same steps were repeated for each of NaOH, SDS, and H 2 O 2 .Finally, the cells washed by 60% ethanol and left suspended in ethanol at 4 ˚C.

S.c.Gs Evaluation Using Light Microscope
Yeast smear for the treated cells was prepared using standard criteria followed by crystal violet stain.The cells were examined by the aid of the light microscope.The quality of the cells was determined based on the yeast 3D structure as either being correct or deformed.

Determination of the DNA Concentration
The concentration of the DNA was determined by measuring the absorption at 260nm.Quartz cuvette was used.An extinction 260 =1 corresponds to 50µg dsDNA mL −1 [22].

Determination of the Protein Concentration
Protein analysis of each experiment (the different supernatants) was determined using the spectrophotometer at 280nm.Quartz cuvette was used.The different protein concentrations were derived from Bovine Serum Albumin standard curve.

Sample Preparation for Electron Microscope Examination
For further study to the yeast, Ghosts quality electron microscope was used to scan the bacterial cells.Dry yeast smear was prepared and the smear surface then coated with approximately 15nm gold (SPI-Module Sputter Coater).

Scanning of the yeast Surface
The golden-coated sample was then scanned by analytical scanning electron microscope (Jeal JSM-6360LA) with secondary element at 10kv accelerating voltages at room temperature.The digital images then were adjusted and saved.

Result and Discussion
This study describes for first time the production of S. cerevisiae Ghosts.The main aim of this study is to prepare S. cerevisiae Ghosts.Recently, Amara et al. [21], have reported a new protocol for Ghosts preparations.The protocol is mainly based on using critical chemical concentration for some chemical compounds are able to introduce genteel pores in the E. coli cells.The original protocol including the use of Plackett Burman experimental design to map the best conditions for obtaining successful Ghosts cells.The protocol was validated in several published papers.The protocol show successful Ghosts preparation from E. coli BL21 and JM109, show sensitivity and prove different MIC between those both similar strains.MIC concentration is a critical point where the used chemical compound kills the cells with the minimum expected damage.The protocol was reduced to be simpler and more practical where the best two experiments obtained from the Plackett-Burman experiment were used only and the MIC and MGC were used to specify the critical point for killing cells without causing any damage.The produced Ghosts from Salmonella typhimurium ATCC 14028 show the sensitivity of such protocol and correct surface antigens maintained during the preparation steps.Such protocol has extended the Bacterial Ghosts preparation, which was prepared using the bacteriophage E lysis gene.It extends its range from only gram-negative bacteria, which restricted due to the function activity of the E lysis gene to nearly all bacterial types.Additionally, Newcastle virus was prepared as Ghosts [23].
Moreover, this study adds the first Eukaryotic cell prepared using such protocol.The Ghosts preparation protocol was rereduced in this study and only MIC concentration of the used chemical compounds were used.NaHCO 3 was used instead of CaCO 3 while it proves to be more powerful against Eukaryotic cells as proved by Amara and Steinbüchels 2014 [21].Yeast cells are bigger than the bacterial cells and they have unique properties that they are able to sediment.For that, centrifugation was not used in this study and only decantation was used.This was planed also, because centrifugation cause Yeasts Ghosts to shrink and their empty inside space when subjected to the centrifugation force which cause the cells to deform or to come together as an empty balloon.As described in the original protocol the Yeast Ghosts DNA and protein losing were determined using the spectrophotometer at 260 and 280nm respectively (data not shown).The amount of the realized protein and DNA show correct evacuation of the yeast cells from their cytoplasmic content as described in the original protocol by Amara et al. [21].The cells with different qualities were monitored using light microscope and electron microscope as in figures 1-3.Both of the light and the electron microscope show correct 3D structure for the prepared yeast Ghosts as in the Figures 1-3.
The study show correct preparation for yeast ghosts could be used in various applications.

Conclusion
This study show for the first time Yeast Ghosts prepared using the critical chemical concentration of the NaOH, SDS, NaHCO 3 and H 2 O 2 .These compounds and by following the concept of the Sponge-Like protocol for preparing Bacterial Ghosts succeeded to prepare yeast Ghosts.Simply, MIC for each has been determined and has been used in steps to induce gentle pore(s) in the yeast cells as well as to evacuate their cytoplasmic contents.The light microscope as well as the electron microscope show correct 3D structure and correct pores in the yeast cells.The spectrophotometer proves correct release for both of the protein and the DNA, which are indications about the evacuation of the yeast cells from their cytoplasmic contents.Such a succeed in the yeast cells preparation open a new area for both drug delivery and vaccination using yeast cells either as carriers for surface foreign proteins or a package can be loaded with proteins as well as for a bigger candidates for loading different drugs and can be used successfully in drug delivery technology.

Figure 1 :
Figure 1: Viable S. cerevisiae cells (before treatment) stained by crystal violet using light microscope.