Do not touch your face when handling plates or tubes with E. Do not put your face near the cultures or the tips when pipetting cultures. Do not incubate plates for more than the given time since this will allow contaminating bacteria and fungi to arise. Follow all directions for post-lab clean-up. Drain excess solution, place materials in plastic bag and dispose in the regular garbage.
DO NOT drip bleach on your clothes as it will permanently stain them. Wipe down lab bench with bleach solution at the end of lab. Wash hands thoroughly before leaving lab. Student Activity: Transformation of the bacterium E. Objectives Understand recombinant techniques and the transformation procedure using the heat shock method.
Understand how we can screen for a gene of interest and the importance of marker or reporter genes in molecular biology experiments. Investigate how DNA can be transferred to another organism and the change in phenotype physical characteristics that may result from such a transfer.
Learn the importance of the sterile techniques that are used to handle bacteria, and the decontamination necessary when the experiment is complete. Procedure Day 1: Make sure that you have all of your group's materials, that you know which other group you will be sharing with, and where the bleach containers are located for clean-up at the end.
Put on gloves and safety goggles. Do not pick up any agar as it may inhibit the transformation process. Exposure of the cells to cold calcium chloride solution, in combination with the "heat shock" discussed in step 12 below, causes the cell membrane to become porous and thus the cells are made "competent" for transformation.
Note: No visible clumps of cells should remain. This step is critical to obtaining good results. Place the loop into the bacterial waste container to kill the bacteria that remain on it. Close the tube lid and put the tube on ice. Both tubes should now be on ice. Your teacher may do this for you. Following the addition of the plasmid, close the tube lid and tap gently with your finger to mix.
Try not to splash the suspension up the sides of the tube. Tap the base of the tube gently on the desktop to make all of the liquid move to the bottom of the tube.
DO NOT add the plasmid to your "-" tube. Incubate both tubes on ice for 15 minutes. Note your observations on the student activity sheet and complete questions Following incubation, "heat shock" the cells. It is essential that the cells receive a sharp and distinct shock.
There were eight non-SNPs in E. The functional classification of COG showed that most genes were related to amino acid transport and metabolism, carbohydrate transport and metabolism, energy production and conversion, general function prediction only, inorganic ion transport and metabolism, and cell envelope biogenesis.
Whole-genome re-sequencing E. The genes in which non-SNPs occur were screened, and E. RT-PCR showed that the orf, orf, orf genes showed high expression in resistant strains E. Notes: A Before the mutation; B after the mutation. Notes: A Before the mutation; B , after the mutation. Before the mutation of orf , 2hrt. A was selected as reference template protein Figure 5A. The model range of residual infrastructure was , the sequence similarity was 0. After the mutation of orf , 1iwg. A was selected as reference template protein Figure 5B.
Before the mutation of orf , 4cti. B was selected as reference template protein Figure 6A. The model range of residual infrastructure was —, the sequence similarity was 0. After the mutation of orf , 3ib7. A was selected as reference template protein Figure 6B.
The model range of residual infrastructure was 10—, the sequence similarity was 0. Regression analysis of MIC and induction time showed that the MIC value of the strain increased with the increase of exogenous antibiotic pressure and the induction time. Liu et al, showed that during the induction of E. Consistent with the results of this study, the MIC value of E.
It shows that if the dose is not limited, the resistance of the strain will become more and more serious. AMP was induced to E. After reaching the critical value, the bacteria may be lazy and grow slowly, but the MIC value continues to increase. It is also believed that this strain activates certain resistance mechanisms and changes the drug resistance status of the bacteria. Zhang et al, showed that chloramphenicol induced sensitive Shigella to the drug-resistant state, and its drug resistance spectrum would change.
Consistent with the results of this study, the drug resistance spectrum of E. The results showed that E. It is considered that during the induction of E. The molecular mechanism of bacterial resistance is still unclear. In order to investigate the specific molecular mechanism of E. The sequencing results were compared with the reference sequence, and 20 SNPs were screened from the sequence of E.
Twenty-six SNPs were screened from the E. Xiang et al, showed that the resistance level of mutant strains was higher than that of non-mutant strains, and there was a quantitative reaction between point mutations and bacterial resistance levels, and multiple gene mutations could enhance the resistance of bacteria to antibiotics.
After sequencing, the non-SNPs screened in this experiment were distributed in the genes of orf, orf, orf, orf, orf, orf, and orf Among them, genes orf, orf, and orf are involved in cell wall synthesis.
The annotations in KEGG are fumarate reductase subunit D frdD , cell division protein ftsI penicillin-binding protein 3 and porin outer membrane protein OmpD, respectively.
Studies have shown that the frd gene encodes a FRD enzyme to catalyze the conversion between fumarate reductase and succinate dehydrogenase.
PBP3 is a core component of cell division proteins that catalyze the cross-linking of cell wall peptidoglycans during cell division. It is considered that these changes in the function of proteins encoded by genes involved in cell wall synthesis affect the resistance of bacteria to AMP. Genes orf, orf, orf are annotated in KEGG as osmotic pressure sensor histidine kinase envZ , multi-drug efflux pump gene acrB , and multi-drug resistance protein involved in transcriptional regulation marR.
In recent years, the active efflux mechanism is the main reason for the multiple drug resistance of bacteria. Cohen et al, demonstrated that the function of the inhibitory protein encoded by the mutated MarR gene would be reduced, and the effect of the bacteria on the multiple resistance of antibiotics was small when the MarR mutation was only detected.
The effector factors can be secreted to the extracellular of bacteria, and the protein secretion system is closely related to virulence of pathogenic bacteria. Wang Jianfeng et al, showed that VgrG gene mutation affects the toxicity and drug resistance of bacteria, but the function of glutamate valine repeat protein is still unclear.
In summary, the COG function of these mutant genes is related to the origin of cell membranes, transport and metabolism of inorganic ions, transcription and signal transduction mechanisms. Studies have shown that under antibiotic stress, bacteria can take both active defense and passive defense to ensure their survival.
In active defense, they increase the activity of efflux pump to increase the efflux of antibiotics and reduce the accumulation of antibiotics in bacteria, thereby reducing the killing effect of antibiotics on bacteria.
This study suggests that the resistance of E. Drug resistance can occur shortly before the bacterial MIC value reaches the drug resistance threshold. These studies will help to improve the molecular mechanism of E. All authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.
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It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Materials and Methods. Audrey Hart , Audrey Hart.
Reprints or correspondence: Dr. Stella Nowicki, Dept. Oxford Academic. Bogdan J. Barbara Reisner. Edyta Pawelczyk. Pawel Goluszko. Petri Urvil. Garland Anderson. Stella Nowicki. Revision received:. Select Format Select format. Permissions Icon Permissions. Abstract The pattern of ampicillin resistance and possible association with virulence factors of 78 Escherichia coli isolates taken from 78 pregnant women with pyelonephritis were evaluated.
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