You have identified a mutation in E. coli K-12 that causes it to become sensitiv

Question

You have identified a mutation in E. coli K-12 that causes it to become sensitive to the the antibiotic azithromycin (azi). You sequence the chromosome of the aziS strain and localize the mutation to genX and near the cysABC operon that encodes the only genes required for cysteine biosynthesis (see diagram). In order to study the impact of the genX* mutation in a pathogenic strain, you would like to move this allele into a uropathogenic E. coli (UPEC) strain background. Design a strategy to move your genX* mutation from E. coli K-12 into the UPEC strain using the strains and plasmids described below. Use point form to describe your strategy. Include a description of all conditions used for selections and/or screens and be clear, but brief about how you will carry out each step. Hint: your answer will require the use of transformation, transposition, and transduction and should include a protocol for a genetic screen for a transposon mutant & the use of generalized transduction to move alleles between strains (9 marks): •Suicide plasmid pMUT: plasmid that encodes the transposon Tn5 & ampicillin resistance; can only replicate at 30oC, not 37oC. the Tn5 element contains a gene that confers a kanamycin resistant phenotype (KnR) •E. coli K-12 genX* : E. coli K-12 strain carrying your genX* mutation that confers azithromycin sensitivity •UPEC wild-type strain •Generalized transducing phage P1, which can infect both E. coli K-12 and UPEC

(all information is given: need to design an experiment with non-mobilizable/ suicide vectors/ transposons/ selection process etc).

UPEC chromosome genX cySA cysB *cySA, B, Care only genes required for biosynthesis of cysteine EC Ampicillin Ori pMUT Tn5 (KnR

Explanation / Answer

1. Transduction and Experimental steps for transduction:-

Transduction is the heritable transfer of bacterial DNA from one cell (donor, here it is E Coli K12) to another (recipient, EColi, UPEC) by a bacteriophage. Tansducing bacteriophage particles are formed in donor bacterial cells during phage development. Generalized transducing phage particles carry a random fragment of host chromosomal DNA approximately the same length as P1 DNA. The phage particles completely lack DNA originating from the phage genome and contain instead oly Bacterila DNA sequesnces. New genotypes in the recipient cells result from homoogous recombination, which can lead to the replacement of a recipient gene by an allele acquired from the donor genome via the transducing phage.

a. Ecoli K 12 (Host cell) infected by phage

b. Host cell DNA broken down into smaller pieces and proteins and phage DNA synthesized

c. Bacterial host DNA is packaged in some viral capsids that are released through lysis of bacterial cell.

d. Transducing phage with host DNA infest new recipient cell.

e. Recombinant new cell contain mixture of donor DNA and its own DNA.

2. Transformation:- It is genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings, through the cell membrane.

Experimental steps for transformation are as follows:-

a. Thawing:- The cells (here UPEC EColi) are thawed on ice and desired DNA (here DNA of EColi K12 with genX mutation) added.

b. Incubation of DNA with cells on ice: For maximum transformation efficiency, cells and DNA are incubated together on ice for 30 minutes

c. Heat Shock:- Both the temperature and the timing of the heat shock are important and specific to the transformation volume and vessel.

d. Outgrowth:- Incubation at proper temperature with shaking and rotating the tubes gives higher transformation efficiency

e. Plating:- Use selcetion plates. Warm and dry plates allow most rapid colony growth.

3. Tranposition:- The ability of genes to change position on chromosomes, a process in which a transposable eleemtn is removed from one site and inserted into a second site in the DNA. The Tn5 transposon system is used for the application of tansposon mutagenesis. Tn5 is a bacterial composite transposon in which genes (antibiotic resistant genes) are flanked by two nearly identical insetion sequences.

Experimental Steps:- The Tn5 transposition process involves following steps

a. Binding of transposase monomers to the end (19bp) sequences

b. Oligomeriztion of the end bound transposase monomers, forming transposition synaptic complex

c. Blunt end cleavage of the transposition synaptic complex from the adjoining DNA, resulting in formation of a released transposition complex or Transposome.

d. binding to target DNA

e Starnd transfer of the transposon 3' ends into a staggered 9bp target sequence. After the strand transfeer, host cell functions remove the trasposase from the product DNA.

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