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Fluorescent labelling of the ER and co-localisation of the DsRed1-E5 protein using confocal laser scanning microscopy
The filamentous fungus Trichoderma Reesei was transformed by harvesting T.reesei from a well-conditioned plate of NaCl and filtered. A very small quantity was plated on a PDA plate and allowed to dry on laminar flow, also some were spread on a PDA plate (positive control) and PDA + Hygromycin (negative control).In the sequel to that, tungsten particles were prepared and were used to shot-in the micro carries particles.After the bombardment, 0.9% NaCl and 0.1% tween was spread on the shot area as an osmotic stabilizer and overlayed with PDA containing Hygromycin at 50Oc. These plates were incubated at 28oc.
The transformant colonies were confirmed by examining the three plates PDA plate (positive control), PDA + Hyg B (negative control) and PDA + Hyg B (transformant colonies) for expected growths. However, colonies from the PDA + Hyg B overlayed plate was restreaked onto a new PDA plate containing cellophane (for easy DNA isolation) and onto a PDA plate containing Hygromycin for second-round selection. The DNA isolation was done by scraping half of the mycelia from the cellophane plate into the mortar, then we added liquid nitrogen to freeze the cell and avoid DNA degradation while we grind to a fine powder in order to break the cell walls and membranes to extract enough quantity of DNA, the fine powder was obtained was placed into an Eppendorf tube and filled up with 2-mercaptoethanol of an aliquot of lysis buffer to break the disulphide bonds between the cysteine residues and removing phenols present in the crude DNA. Chloroform and Phenol were also added to achieve a clear separation between the aqueous and organic phase layer making the aqueous phase easy to be removed to obtain a pure sample. Also, during the process, we used Sodium acetate to increase the ion concentration in the solution for easy precipitation by the Isopropanol. Furthermore, we washed the DNA pellet with 70% ethanol to get rid of the excess salt which dissolves in 30% of the water and also allows the DNA to retain some water molecule as 100% ethanol will leave the DNA wholly dehydrated and insoluble. Afterward, the quality and concentration of the chromosomal DNA were checked using the Nanodrop spectrophotometer, which showed that I had good DNA but was contaminated with phenol, alcohol, and salts which might be due to not properly washing with enough ethanol. We went further to identify the size of the DNA and also check RNA presence in genomic DNA through gel electrophoresis, the DNA was separated to different bands which the sizes was ascertained by comparing to the molecular marker, but the DNA was degraded due to some contaminations as there was no distinctive band shown. Then, the transformed T. reesei expressing the DsRed 1-E5 protein on the pHEN54RQDsR plasmid under the cbh 1 locus was amplified using a complementary pair primer DsRedprobe.fwdpr and DsRedprobe.rvdpr and to confirm the presence of DsREd-E5 gene in the genome of T.reesei, the sample was stained with GedRed and visualizing the PCR product, we have a band of approximately 200bp which confirms the presence of the transformed gene sequence on the fungi sequence. The successful transformant was now visualized on a confocal microscopic laser scanning microscope to check the expression of the DsRed1 protein.
Fluorescent labeling is the process of covalently binding fluorescent dyes to biomolecules like nucleic acids or proteins so that they can be visualized by fluorescence imaging. However, fluorescent probes are designed to localize within a specific part of the cell such as Endoplasmic, cytoskeleton mitochondria, and nucleus. Fluorescence is then used as image mode due to its high sensitivity and targeting of structural components in both fixed and living cells and tissues. Fluorescence microscopy is a very crucial tool for interpreting the cellular functions of proteins and some other molecules. Mostly molecules can be predicted from its association with intracellular complexes, which is ascertained from comparing the fluorophores with the others been investigated.
Confocal laser scanning microscopy (fig 1) is a method that is more of optical imaging which boosts evaluations of a micrograph together with an optical resolution with the use of a Pinhole to shed from the focus of light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures within an object.
Fig 1 Laser scanning confocal microscope
Aims: This experiment enhances our understanding of the capabilities of CLSM,
Visualization of the expression of DsRed1 protein in T.reesei transformed with Phen54qdSr plasmid (fig 2) using a confocal laser scanning microscope coupled with understanding based on fluorescent spectroscopy and imaging.
Materials and Methods
Table 1: Laser types
ER –TrackerTM Green
The culture (transformant) was prepared by adding 1% (w/v) Agarose to CLS (Cellobiose-Lactose-Soy hydrolysate; pH 5.5) medium and prepared according to Lim et al. (2001) procedures. The agarose medium was placed in six wells culture dish and was allowed to cool. Also, the pre-sterilised cellophane disk was washed with molten 1% (w/v) agarose to form a thin layer of coating of the cellophane and was placed on the culture dish. Furthermore, the conidial suspension of each T. reesei strain was used to inoculate the central point of the disk, and the cultures were incubated at 28ºC in the dark for 48 hours. After the incubation, the cellophane disk was carefully lifted using tweezers from the culture dish and was placed on paper inside a petri dish. Then, we used razor blade to cut the disk into half, cutting through the canter of the colony, a small wedge was gotten, which was floated keeping the hyphae-side down onto a drop of stain dropped at the marked centre of the slide, ER-Tracker™ Green (1 µM in PBS),on the already prepared slide. Afterward, we stained for 30 minutes in a black incubator for proper binding and the stain was drawn off. In the sequel to that the wedge of cells on the slide was washed thoroughly with 1*PBS and was taken back to the incubation chamber ready for confocal microscope fluoview.
Result and Discussion
I ii iii
Fig 4 (W18)
iv v vi
Considering the fig 3 above, the expression of gene Dsred1 E5 in T.reesei transformed with fig 2, it was detected using the two lasers from table 1 and overlap of both lasers and images I, ii and iii was visualized. Image (ii) and (iii) shows the sparse distribution of DsRed1 protein and ER in the hyphae, also some spots observed with both lasers in ii and iii are similar which is a very high chance of overlap between the two fluorophores at the same location in the hyphae (co-localisation). However looking at the image (i) above the apical hyphae has a high intensity of yellow spots which shows that both the DsRED1 and ER were present while in the apical hyphae only green spots were seen, which inferred that there was no enough protein at the tip.
Investigation (fig 4) above, iv, v and vi has a well randomly distributed fluorescence spots with a high intensity unlike what we saw in fig 3, similarly to what we explained in fig 3 the images from iv and v are similar which also instigate co-localization. Then looking at image vi its shows that the major cells have both DsRed1 and ER presents but had more of DsRed1 than the ER at the tips as some red spots are located around the apical and in some n0n-apical part of the hyphae.
A red fluorescent reporter protein is very essential for fluorescence microscopy in T.reesei as is not autofluorescence in wavelengths 504 – 583. In fig 3, the concentration of both the DsRED1 and ER are same in the major cells of the hyphae which infers that both are same proportion in mature cells as in the secretory pathway the ER is already embedded in the hyphae while some time is allowed for the protein synthesis whereas, the concentration of red is low compared to the green concentration at the tip of the hyphae. Also looking at the control (Rut-C30) there was no fluorescence observed using both lasers.
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