Due to system limitations you are attempting to clone a small bacterial gene using a poorly...

Due to system limitations you are attempting to clone a small bacterial gene using a poorly characterised bacterial plasmid. You know that the plasmid contains only two drug resistance markers (one for resistance to tetracycline, another for resistance to ampicillin). You have determined that the restriction enzymes EcoRI and BamHI each cut the plasmid only once while SauIII and RsaI cut the plasmid twice. You also have determined that the EcoRI and SauIII recognition sites are within the tetracycline resistance marker, but the BamHI and RsaI recognition sites are not within or even close to either drug resistance marker. You must use this particular vector: a. Which restriction enzyme(s) should you use and why? b. What will happen if you use the BamHI recognition site? What will happen if you use the RsaI recognition site? (Describe this with regards to the functioning of the drug resistance marker and which antibiotic(s) you should use in the media)

Solutions

Expert Solution

Plasmid map assuming all the information has been shown below

As EcoRI and BamH1 cut the plasmid once

And Sau II and Rsa II cut DNA twice

And their sites are ECORI and Sau III cut with in Ter resistance gene.

While BamHI and RsaII cut near to ampicillin resistant gene.

Image has been attached below, this image is made just based on given information to understand

A) to use a restriction enzyme site for insertion of a gene

Its site must be in antibiotic resistant gene

And it should have only one RE site or ( single cut).

Based on the information, it is the EcOR I which can be used for cloning purposes. As BamH1 and Ras II do not cut in antibiotic resistant gene.

Second Sau II cut twice in Plasmid DnA . So ECoR I is most appropriate enzyme to use in cloning.

B) these antibiotic resistance gene used for selection of plasmid when grow in culture media.

If we use these two enzyme BamH1 and Ras I

BamH1 as it has single RE site so it will lead to retain of both amp and ter resistance enzyme and selection can not be made.

If we use RsaI than as it cut DNA twice so it may lead to loss of a big part of plasmid which may be essential for plasmid replication or some other process

This process has also been shown in above attached image.

Hope this will help .if you have any query than let me know

gladiator answered 1 year ago

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