التفاصيل البيبلوغرافية
| العنوان: |
Development of tools for intracellular pH measurement in yeast and fungi ; Hiivan ja homeiden solunsisäisten pH-arvojen mittausmenetelmien kehitys |
| المؤلفون: |
Kaija, Jenni |
| المساهمون: |
Valkonen, Mari, Kemian tekniikan korkeakoulu, School of Chemical Engineering, Biotekniikan ja kemian tekniikan laitos, Leisola, Matti, Aalto-yliopisto, Aalto University |
| سنة النشر: |
2011 |
| المجموعة: |
Aalto University Publication Archive (Aaltodoc) / Aalto-yliopiston julkaisuarkistoa |
| مصطلحات موضوعية: |
intracellular pH, saccharomyces cerevisiae, trichoderma reesei, fluorescence microscopy, spectrofluorometry, ratiometric probe, pHluorin, D-xylonate, L-galactonate, solunsisäinen pH, fluoresenssimikroskopia, spektrofluorometria, ratiometrinen koetin, RaVC, D-ksylonaatti, L-galaktonaatti |
| الوصف: |
The aim of this work was to optimize tools for intracellular pH (pH i ) measurement in yeast Saccharomyces cerevisiae and filamentous fungus Trichoderma reesei using genetically encoded ratiometric pH probes, pHluorin and RaVC. The pH probe was expressed in control strains and strains that are known to produce different acids in the culture medium: in a S. cerevisiae D-xylonate producing strain, a T. reesei L-galactonate producing strain and a T. reesei 2-keto-3-deoxy-L-galactonate producing strain. Measurement of the pHi was done with living cells using two in vivo methods: imaging and measuring the cytoplasmic pH of single cells using a standard fluorescence microscope (FM), and measuring the cytoplasmic pH of the cell populations using a spectrofluorometer (SFM). It has been shown that the acids accumulate in the cytoplasm and in this work it was studied, if there were differences in cytoplasmic pH in acid producing and control strains. The method measuring pH i with the FM could be optimized to the level that the pH could be measured. Better accuracy was achieved with the spectrofluorometric method which worked well with S. cerevisiae strains. The pK a value of RaVC measured with the FM was 7.0±0.2. The pK a value of pHluorin measured with the FM was 7.3±0.1 and with the SFM 7.1±0.1. The excitation maxima of pHluorin were measured with the SFM to be 395 and 475 nm. The pH i values of the strains were measured without inducing acid production. The pH i of the S. cerevisiae control strain was 6.4±0.5 and the pH i of D-xylonate producing strain was 6.6±0.5 when measured with the FM and respectively, 6.9±0.2 and 6.9±0.1 when measured with the SFM. The results indicated that there are no differences between the strains when grown under conditions where there is no intracellular accumulation of D-xylonate in the D-xylonate producing strain. The pH i of the T. reesei control strain was 7.0±0.3 when measured with the FM. The pH i of T. reesei has not been published earlier. Other constants and pH i values ... |
| نوع الوثيقة: |
master thesis |
| اللغة: |
English |
| العلاقة: |
https://aaltodoc.aalto.fi/handle/123456789/99337Test; URN:NBN:fi:aalto-2020122358164 |
| الإتاحة: |
https://aaltodoc.aalto.fi/handle/123456789/99337Test |
| حقوق: |
closedAccess |
| رقم الانضمام: |
edsbas.30172AEF |
| قاعدة البيانات: |
BASE |
