Browsing by Subject "ddPCR"
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(2022)Severe acute respiratory syndrome coronavirus 2 is the pathogen behind COVID-19, a contagious disease that has caused millions of deaths since the beginning of the global pandemic in 2019. It is essential to gain more knowledge about the factors affecting viral entry and infection to understand prevention and treatment possibilities for the pathogen. Initially, an immunostaining assay was set up and showed that at eight hours post-infection, the virus had entered the target cells and started protein production. Five host genes of interest, identified by collaborators, were transiently silenced using small interfering RNA knockdown of cell lines. Once the silencing had been optimized, the effect on SARS-CoV-2 infection was monitored at eight and 24 h post-infection. The main pipeline used to follow the cell entry and replication was an extraction of total RNA, reverse transcription reaction followed by quantitative digital droplet polymerase chain reaction, showing the concentrations of the expressed target genes in the cells. The protocols of the transfection and ddPCR were tested and optimized to provide reproducible and reliable results. Out of six tested transfection reagents, DharmaFECT 2 showed the most effective results, decreasing the expression of target genes DDX3X and PDE4A by more than 75% after a 24 h incubation. The produced knockdown cells were infected with a produced and purified stock of SARS-CoV-2. Plaque assays were made from samples collected 24 hours post-infection, but no difference could be seen in the titer of the virus between the transfected cells and negative control cells. In conclusion, using digital droplet polymerase chain reaction to follow the effect of host gene knockdown on virus replication is possible as long as the initial gene expression is high enough.
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Surface layer protein SlpC expression in different growth modes of Lactobacillus brevis ATCC 14869 (2020)Bacterial surface layers (S-layers) usually consist of a single protein or glycoprotein species and they form the outermost layer of many bacteria. Three S-layer protein genes have been found in Lactobacillus brevis ATCC 14869, but the expression of only two of them, slpB and slpD, has been described in planktonic conditions. Preliminary results have suggested that the third gene, slpC, may be expressed in biofilm cells. The aim of this thesis was to study the expression of S-layer protein genes slpB, slpC and slpD in the different growth modes of L. brevis ATCC 14869. Surface proteins were detached both chemically for SDS-PAGE analysis and enzymatically for surfaceome analysis by LC-MS/MS and label-free quantification (LFQ). Messenger RNA transcript levels of the S-layer protein genes were analyzed by reverse transcriptase droplet digital PCR (RT-ddPCR). The expression of all three S-layer protein genes slpB, slpC and slpD was confirmed in the study. In the surfaceome analysis, SlpD was the most abundant protein detected on the cell surface, representing more than a third of all identified proteins, whereas SlpB and SlpC were less abundant. The S-layer proteins were detected by gel electrophoresis. Transcripts of all three genes were observed; slpD transcript amounts were the highest, whereas slpB and slpC transcript amounts were lower. The S-layer proteins and their transcripts were present in equal amounts in biofilm and planktonic cells. In this study, SlpC expression is demonstrated for the first time in L. brevis ATCC 14869. The expression of slpB, slpC and slpD was at the same level in biofilm and planktonic cells.
Now showing items 1-2 of 2