Question

Describe how you would identify genes responsible for transcription in E. coli by isolating mutations in their genes. Remember that these genes are required for cell viability. Include all the steps in the isolation of the mutants and also describe how you will differentiate the mutants you want from those in other genes necessary for viability. This includes giving me the expected result for mutants in the genes you want.

Answer 1

So, here’s the answer to your question.

Let us start with the main part of the question that is the steps that are followed in the isolation of mutants.

The process of cloning, say for a particular DNA segment. There are enzymes for the restriction process to happen that is the restriction enzymes. After this process, there is ligation and also the transformation of a gene. This entire process is well described as four simple steps. Let me list it for you..,

• As mentioned the isolation of any DNA as per the requirement
• Next is the ligation
• the following ligation is the transformation
• and the final is the process of selection

The most widely used restriction enzymes include the EcoRI, Sau3A,TaqI etc

Moving onto the second most important part of the question that is the differentiation aspect. This is done by the process called the Gel electrophoresis. This method is used for the separation of the DNA fragments based on the charge as well as the size of the fragments. Using the gel which usually made of the substance like gelatin composing of either the agarose or sometimes the polyacrylamide depending on the requirement. Then on the one end of the gel, there is an application of an electric field with a positive charge and the negative charge is applied on the other side of the end. This results in the movement of the fragmented DNA. The phosphate groups are the negatively charged move towards the positive charge. The molecules which are long in size take a little more time to move when compared to the smaller ones. The separation comes to an end after this process. This process can be observed using a fluorescent dye that is suitable to the DNA taken. It is the ethidium bromide which is commonly used. The gel when stained gives a better picture of the DNA molecules of different lengths as well as the molecular weights. DNA ladders are used for the comparison with reference to the known length DNA fragments

I hope this helps. Please do let me know in the comment section if I can help you with anything.

BEST OF LUCK

Thank you

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