The transposon Tn5 has been widely used to create Escherichia coli (E. coli) str
ID: 93217 • Letter: T
Question
The transposon Tn5 has been widely used to create Escherichia coli (E. coli) strains having knock-out mutations in specific chromosomal genes. A suicide delivery plasmid vector that carries Tn5 is used for this purpose. Explain why a suicide delivery plasmid is used, and how you would use this plasmid to obtain E. coli mutants with Tn5 insertions in specific genes of interest, for example in the lac (lactose) operon. The transposon Tn5 has been widely used to create Escherichia coli (E. coli) strains having knock-out mutations in specific chromosomal genes. A suicide delivery plasmid vector that carries Tn5 is used for this purpose. Explain why a suicide delivery plasmid is used, and how you would use this plasmid to obtain E. coli mutants with Tn5 insertions in specific genes of interest, for example in the lac (lactose) operon. The transposon Tn5 has been widely used to create Escherichia coli (E. coli) strains having knock-out mutations in specific chromosomal genes. A suicide delivery plasmid vector that carries Tn5 is used for this purpose. Explain why a suicide delivery plasmid is used, and how you would use this plasmid to obtain E. coli mutants with Tn5 insertions in specific genes of interest, for example in the lac (lactose) operon.Explanation / Answer
Suicide delivery plasmid vector are those plasmids which are replication incompetent. This means that the plasmid has got its sequence lacking the "origin of replication" or the ‘ori’ site. This loss makes the plasmid incompetent to take part during the replication at the time of cell division, and eventually gets diluted from the cell in which it is inserted, after few generation. Finally we can say that from the population of DNA molecules inside each cell it promptly becomes eliminated by "committing suicide".
This logical explanation is important to explain the Tn5 transposon activity in the cells of E. coli. In gene knockout technique we can make an organism gene inoperative. Now using this suicide delivery we can put our vector construct into a bacterium where the transposon gets transferred and the unwanted part of the vector will get dissolved. So here using this suicidal plasmid our purpose is getting served.
For vector insertion into a bacterial cell, we can use methods like ‘Heat Shock technique’ or microinjection, thereby once the vector is inside the bacterial cell we can transfer the Tn5 transposon. We will look for the restriction enzyme sites in lac operon and the same sequence of restriction we will put before and after our transposon construct. In this manner the jumping gene or transposon after getting inside a cell will go and insert itself into the site of lac operon.
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