Browsing by Subject "electroporation"

Protein-protein interactions (PPIs) regulate many different cellular processes including transcription, translation, cell division, signal transduction, and oncogenic transformation. It is therefore important to develop sensitive and versatile techniques for the detection of these protein-protein interactions in order to fully understand protein functions. The most commonly used and traditional technique, the yeast two-/three hybrid (Y2H/Y3H) method, often results in false positives and false negatives, and other widely used techniques, such as bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), and bimolecular fluorescence complementation (BiFC) require extensive instrumentation. When compared with other PPI detection methods, the luciferase-based complementation assay specially split luciferase is believed to deliver the most sensitive and highest dynamic range, making it ideal for large-scale analysis. Therefore, for testing PPIs in planta, split Renilla luciferase complementation assay was chosen. In order to conduct this experiment, a series of plasmid constructs were made to enable the transient expression of fusion proteins. A well known protein pair, Arabidopsis nuclear Histone 2A and 2B, was tested initially as a proof of concept, and then three more proteins of the Gerbera MADS-box B class were investigated. For Arabidopsis Histone 2A and 2B, the intensity in all combinations was on average 9.4-fold higher in Relative Luminescence Units (RLUs) than the mock treated protoplasts. Moreover, in the case of Gerbera MADS-box B class proteins, the protein pairs GDEF1-GDEF2, GDEF1-GGLO1, and GDEF2-GGLO1 showed 8.4-19.4, 9.5-15.8, and 8.3-9.1-fold higher signals than the mock treated protoplasts. These results suggest that various complexes formed from different combinations of these three B class MADS-box proteins may increase the complexity of their regulatory functions, thus specifying the molecular basis of whorl morphogenesis and combinatorial interactions of floral organ identity genes in Gerbera. Finally, it was concluded that split Renilla luciferase can be a simple, reliable, fast, and effective method for examining PPIs in planta.

Bacteriocins are ribosomally synthesized antimicrobial proteins. They can be applied as biopreservatives in food processing for extending the shelf-life of food. Lactic acid bacterium Leuconostoc carnosum 4010 is Generally Recognized As Safe strain, which can be used as a protective culture in meat products. The strain 4010 produces three bacteriocins: leucocins A (LcnA), B (LebB) and C (LecC). For the secretion of bacteriocin out from the cell, bacteria usually use an ABC transporter, which is often dedicated to secrete only one bacteriocin. The leucocin operons in Ln. carnosum 4010 plasmids include genes for only one ABC transporter, namely LecXTS. The fact that Ln. carnosum 4010 produces three bacteriocins but only carries one bacteriocin transporter, raises a question, which leucocin(s) is/are translocated via LecXTS transporter. Therefore, the first aim of this study was to determine which bacteriocin(s) is/are secreted by LecXTS in Ln. carnosum 4010. Ln. carnosum 4010 carries at least two plasmids. Leucocin A gene lcnA is located on the plasmid pLC4010-2, and leucocin B and C with transporter genes (lebB, lecC, lecXTS) are located on the plasmid pLC4010-1. In a previous work, two plasmid cured derivatives of Ln. carnosum 4010 have been made: the plasmid-free strain PCS-10, and the strain PCS-11 carrying only pLC4010-2. Neither of the derivatives secrete bacteriocins. In this study, the idea was to construct five recombinant plasmids containing the pLC4010-1 replication gene repB and a gene for erythromycin resistance ErmR. They were ligated with different sets of leucocin and transporter genes (repB-lebB-lecXTS-lecC-ErmR, repB-lebB-lecXTS-ErmR, repB-lebB-ErmR, repB-lecC-ErmR, and a vector control with only repB-ErmR). The constructs were aimed to be introduced into the two Ln. carnosum 4010 mutant strains PCS-10 and PCS-11. However, after several attempts of electroporation, no colonies were obtained. To acquire a testing plasmid for optimization of transformation, the ligation mixture for the smallest plasmid repB-ErmR was electroporated into another strain, Lactococcus lactis N8. The plasmid repB-ErmR was successfully obtained from Lc. lactis N8. For improving the efficiency of transformation, the plasmid repB-ErmR was isolated from Lc. lactis N8, and the plasmid was used in optimization of electroporation. The copy number of the plasmid was shown to be very low, as only a little amount of plasmid could be isolated from large culture volume. Even with optimized electroporation method, the repB-ErmR could not be electroporated into Ln. carnosum 4010. This indicates that the larger constructions are nearly impossible to be transferred into the strain Ln. carnosum 4010. In conclusion, it was confirmed that the plasmid replication gene repB of Ln. carnosum 4010 is functional in Lc. lactis. Due to the low copy number of the plasmid repB-ErmR, the amount of plasmid was definitely a problem in electroporation. Therefore, for studying the efficiency of electroporation, the plasmid amount needs to be increased. Although the electroporation of repB-ErmR into Lc. lactis was successful, the results from Ln. carnosum electroporation after optimization indicate that the strain Ln. carnosum 4010 is difficult to be transformed.

Background and objectives: Cells secrete extracellular vesicles (EV) and it has been found that cells communicate via EVs. EVs are liposome-like vesicles. Membrane is consisting of a lipid bilayer and hydrophilic moiety is inside the vesicle. It has been found that EVs carry e.g. nucleic acids, lipids and proteins. The aim of this master thesis was to determine whether EVs can transport non-coding RNA (siRNA) into the central nervous system through the blood-brain barrier. In the literature review, investigated methods which has been used to load siRNA into the EVs and how EVs are transported through the blood-brain barrier. The aim of the experimental part was to produce and isolate EVs and to load FAM-labeled dsDNA and siRNA into EVs by physical methods such as sonication and electroporation. Fluorescence measurements were taken to demonstrate FAM-labeled DNA loading into EVs and the functionality of the siRNA-loaded EVs was measured by measuring the expression level of the gapdh gene. Methods: Extracellular vesicles were produced in ARPE-19 and PC-3 cells. EVs were isolated from the cell culture medium by two-step differential centrifugation (DC) and further purified by gradient centrifugation (GC) by using the OptiPrep™-reagent. OptiPrep™-reagent was purified by Amicon 10kDa filtration tubes. The average particle size and size distribution of the isolated EVs were determined by NTA analysis, protein concentration was measured by colorimetric BCA method and EVs were characterized by Western blot method using HSP70 and CD9 antibodies. EVs were loaded with 21 bp length FAM-labeled dsDNA or siRNA by sonication or electroporation. Free nucleic acid and OptiPrep™-reagent were purified from EVs by the size-exclusion chromatography with Sephacryl (S-300) column. Loading efficient of the EVs were studied by measuring the fluorescence (ex 485 nm, em 520 nm) and qPCR method was used to demonstrate the functionality of the siRNA loaded EVs. In qPCR, the expression level of the gapdh gene was measured in dividing ARPE-19 cells. Results: DC and GC purified ARPE-19 and PC-3 EVs had an average particle size of about 140 nm and were successfully characterized by Western blot method. PC-3 EVs were produced in the bioreactor and the yields were enough for loading experiments. ARPE-19 cells produced only small amounts of EVs in culture flasks. The size-exclusion chromatography was a good method to purification free nucleic acids from EVs. The sonication method did not cause EVs to be degradation under the conditions used. Based on fluorescence measurement, FAM-labeled dsDNA could not be loaded into EVs. The functionality of siRNA-loaded EVs could not be demonstrated in ARPE-19 cell experiments. After electroporation large number of EVs were lost and this method of loading siRNA into EVs did not proved to be suitable. Conclusions: ARPE-19 EVs must be produced in the bioreactor to produce enough EVs for loading experiments. The EV purification protocol should be further optimized since the recovery-% of EVs were low after several purification steps. The size-exclusion chromatography is suitable for the purification of the free siRNA from EVs, but the chromatography method needs further optimization and miniaturization. Loaded EVs should be produced by aseptically or alternatively sterilized prior to ARPE-19 cell assay. Physical loading method, such as sonication, can be scaled to larger scale. Sonication method should be optimized e.g. by experimenting with higher temperatures and longer sonication times. The probe sonicator should be tested instead of the water bath sonicator. According to the literature review, the use of extracellular vesicles as carriers for biomolecule delivery into the central nervous system seems to be promising.