Techniques | K610-MUSC

Research Techniques

Our laboratory facilitates the BSL2 laboratory system due to Pseudomonas aeruginosa, a human-pathogenic bacterium, as our biological microorganism study. We apply the DNA recombinant techniques and use genetic engineering tools to construct the mutant strains in order to delete or to overexpress the specific gene of interest. To confirm the mutation of gene in the P. aeruginosa mutant chromosome, PCR, DNA sequencing and Southern blot analyses will be required. Moreover, various physiological analyses against stress-mediated conditions including plate sensitivity, oxidant-killing, and disk-diffusion assays will be done to determine the level of stress tolerance and to compare with the wild-type strain. In order to investigate the cis-regulating region or promoter region of gene, either primer extension or 5'RACE techniques will be set up. The gene expression level will be interpreted by analysing the mRNA level of that gene using several techniques such as hot-acid phenol chloroform RNA extraction, DNA or RNA bioanalysis, Northern blot, end-pointed RT-PCR, and real time RT-PCR. The function of gene product are also in our observation. Thus, several enzymatic activity assays such as beta-galactosidase, catalase, superoxide dismutase, aconitase, nitrate reductase, succinate dehydrogenase, and cytochrome C oxidase activities will be performed by comparing the activity of crude protein from different bacterial cells. Protein expression and purification of several forms (native and site-directed mutagenic) of protein will provide us an in vitro binding experiments. Gel electrophoresis mobility shifted assay (EMSA) and foot-printed analysis will be performed to investigate the binding site of many transcriptional regulators on several gene promoter regions. We apply Drosophilla melanogaster and Caenorhabditis elegans host model systems for testing P. aeruginosa pathogenicity and determine the virulence level.

Solution Preparation

Medium Preparation

Bacterial Growth

Chromosome Extraction

Plasmid Extraction

Transformation

Blotting Hybridization

RNA Extraction

Gel electrophoresis

Site-directed mutagenesis

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Genetic modifications

Cre-loxP deletion system

Knock-out system

Plasmid/Tn7 insertion

Site-directed mutagenesis

Physiological studies

growth inhibition

treatment-killing

virulence assays

To construct bacterial mutant strains, several techniques will be selected due to the propose of analysis. For example, Cre-loxP deletion system are used when we would like to delete a specific area of DNA on the chromosome, normally in the gene of interest. This technique is able to get rid of antibiotic marker after gene deletion, which facilitates us to determine an antibiotic profile of the mutant without cursion. In order to change some amino acids in the protein expression plasmid, we generally use PCR-based site-directed mutagenesis tools for constructing the plasmid that exhibits the change in bases and leads to alter amino acid of interest.

After strain construction, we compare the physiological changes in several phenotypes including growth under oxidant or antibiotic exposures. Plate sensitivity and disk-difffusion assays are primarily screen the change of phenotype comparing between the wild type and the mutants. These results indicate the plate growth inhibition effect of oxidants or conditions treated. We also observe the killing rate of these treatment on these bacteria in liquid broth. This technique is done by direct killing bacteria and observe the survivals. Moreover, we apply Drosophilla melanogaster and Caenorhabditis elegans host model systems for testing P. aeruginosa pathogenicity.

Gene regulations

promoter identification

gene expression

real time RT-PCR

EMSA, foot-print analysis

In order to investigate the gene regulation, we has to analyse the promoter region of the gene of interest. Several techniques are applied such as primer extension or 5'RACE to identify the transcription start site and furhter determine putative RNA olymerase binding regions (-35 and -10). Regulation of gene is devided into two mechanisms, direct and indirect. Direct regulation is that the regulator directly affect the promoter region of the gene and influent the gene expression. Up-regulation is to increase the gene expression and the regulator acts as an activator while down-regulation is to decrease the gene expression and the regulator acts as a repressor. Normally, these regulators bind to the DNA in the promoter region and affect the transcriptional initiation. Indirect regulation is to regulate via an intermediate, which can be another protein or other sRNA.

Enzymatic assays

protein purification

protein gel assays

spectrophotometric

chemiluminescent

To determine the activity of enzymes, we can devided into two methods. First we measure the enzymatic activity from the crude protein of the cell samples, which are from different strains. This is much more like the level of enzyme and the activity in the cell due to we can interpret to the activity that occurs inside the cell and it faces less problem about co-factors of the enzymes and required enzymes in the reaction pathways. Another method is to express the proteins, to purify the proteins, and to set up the reaction of catalysis. This is suitable for the proteins that does not require rare cofactor or not need specific condition. Some proteins easily degrade when exposure to oxygen, this will make us in trouble to do the second method. To observe the enzymatic reaction, we use various types of detections depended on the propose of analysis.