Study of bacterial DNA compaction after slow lysis of spheroplast | |
| Author | Shrestha, Pravin Malla |
| Call Number | AIT Thesis no.BT-03-15 |
| Subject(s) | DNA Spheroplasts Bacterial cell walls |
| Note | A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science, School of Environment, Resources and Development |
| Publisher | Asian Institute of Technology |
| Series Statement | Thesis ; no. BT-03-15 |
| Abstract | The genetic material DNA is stored in the cell with degree of utmost compaction. In bacteria, the DNA molecule has a length of about 1500μm with an internal diameter of around lnm. In this research spheroplast have been prepared with an aim to observe the release of their DNA during a mild cell lysis procedure. Selected strains of the bacterium E.coli were cultured in LB medium containing different concentrations of ampicillin antibiotic. Cell in the cultures were converted into spheroplast by treatment of different dilutions and pH of lysozyme buffer. The best strain was selected on the basis of the recovery time of the spheroplast. Then the isolated spheroplasts were treated with different solutions including sucrose, water, 0.1 % SDS, 1 % SDS, 1 OmM CT AB buffer and 70% ethanol. Spheroplast lysis and the nucleoid compaction were studied by using phase contrast microscope and fluorescence microscope after staining the DNA with DAPI and ethidium bromide. The slowest lysis procedure was used for the lysis of the spheroplast and observation by Scanning Electron Microscopy. It was found that the spheroplast isolation time was reduced many folds when an ampicillin-containing medium was used for the culture of the organism. The concentration of 0.4μg/ml of ampicillin in the medium was the best for the isolation of the spheroplast. The best strain for the isolation of the spheroplast was found to be E.coli wild type, the buffer was tris or phosphate buffer pH 8 and the lysozyme concentration was 2mg/ml. The nucleoid was found to be reduced in size (reduced in diameter), when lysis was achieved in high concentration sucrose solution and 70% ethanol. Instead the size of the nucleotide was found to be increased in diameter initially and later it was bursting, when water or detergents were used. SEM sample preparation was done on membrane filter and on cover slips. During sample preparation it was learned that the sample preparation on the cover slip was more reliable, easier and accurate. The DNA visualization after the lysis of spheroplast was not conclusive under the SEM for the same we need to apply Atomic Force Microscopy or cryo electron microscopy. |
| Year | 2003 |
| Corresponding Series Added Entry | Asian Institute of Technology. Thesis ; no. BT-03-15 |
| Type | Thesis |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Food, Agriculture and Natural Resources (Former title: Department of Food Agriculture, and BioResources (DFAB)) |
| Academic Program/FoS | Food Engineering and Bioprocess Technology (FB) |
| Chairperson(s) | Stevens, W.F.; |
| Examination Committee(s) | Yang Yi;Sunee Nitisinprasert;Suwalee Chandrkrachung; |
| Scholarship Donor(s) | The Government of Austria; |
| Degree | Thesis (M.Sc.) - Asian Institute of Technology, 2003 |