Investigation of the effect of the chemotherapy agent doxorubicin on escherichia coli and the genes related to resistance by genome-wide library and screening studies

Cancer cells may occasionally develop resistance against chemotherapeutic drugs used in the treatment of cancer diseases. The cancer cell can escape from the drug by changing the structure and expression of some genes, and with cancer recurrence, fatal results can be seen. The identification of the genes and proteins that may be involved in the molecular mechanisms of resistance is important both in terms of basic science and in terms of offering treatment options. Doxorubicin is a common chemotherapeutic agent that is routinely used in the treatment of cancer, but also has an antibacterial effect. When this drug is given to a cell, it damages DNA and can cause the death of the cell. However, there are reports that the cancer cell can also develop resistance to this drug. The determination of the molecular targets of the drug and the identification of the genes that have a direct or indirect role in the development of resistance should be investigated in different cellular systems. The aim of this thesis study is to find the gene candidates that provide resistance to the cell, and to achieve this, 4123 genes will be screened at increasing doxorubicin concentrations by using the bacterial cell as a model. Here, the ASKA clone set obtained by cloning the genes of Escherichia coli into an expression plasmid was used. This set, composed of 57 microplates, is a unique genomic source that allows to examine the relationship between the drug and 4123 genes in a different manner. Since the expression of each gene can be induced by IPTG, screening can be conducted at various expression levels. The approach is based on the following criteria. The clone that survives at the drug concentration in which the wild type dies is determined and the gene within the particular clone is considered to be related to resistance phenomena directly or indirectly. In this study, first, the preliminary experiments with several different E coli wild-type bacterial strains were conducted and the concentrations that have bactericidal effect on the strains were determined using minimum inhibitory concentration (MIC) experiments, growth curve experiments, spot tests and disc diffusion methods. Then, "genome-wide plasmid library" of 4123 clones was transformed into E. coli D10Hb strain by the heat shock method. Using concentration data obtained from optimization experiments, selection experiments were carried out with gradient cultivation, direct cultivation and enrichment in order to identify the clones that can live in environments with relatively high doxorubicin. Validation studies were carried out with 129 colonies that had doxorubicin tolerance. Then 34 colonies with the highest tolerance were selected. The gene sequences contained in the selected recombinant plasmids were determined by Sanger sequencing analysis. Using the sequences obtained, gene information was found by Blast analysis. As a result of the studies, it was determined that the genes in the plasmids of E. coli clones living at relatively high doxorubicin concentrations were recA (n = 23 clones) and mdtK (n = 11 clones). It has been discussed that these genes are involved in repairing DNA damage and expelling the drug out of the cell using Na+ channels. In addition, homologs of these genes in the human genome were searched. This thesis study, approaching the topic of cancer from a different angle, where the bacterial cell is used as a model system has successfully analyzed the relationship between thousands of genes (n=4123) and a drug using molecular biology, microbiology and genomic disciplines and ended up in producing comprehensive, original and guiding results. Key words: Doxorubicin, Escherichia coli, gene, genomics, mdtK, recA, resistance