Identification of reporter genes

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Introduction

Reporter genes are genes with particular characteristics that make them easily identifiable when they are expressed by the organisms to which they are attached.  They are used to determine if a gene of interest has been expressed within a cell.  As a result of which they have become an  invaluable tool in research for the areas of biochemistry, molecular biology, biomedical and pharmaceutical sciences.

In the case of this paper the following reporter genes were used to identify the following organisms:

In order to introduce the reporter gene into an organism, the reporter gene and the gene of interest are inserted into a circular DNA strand known as a plasmid (vector). Here we used the following plasmids; pUCD607, pUC18 and the suicide vectors FAC510 and pUT-miniTn5/xylE so called because they cannot survive once integrated with the bacterial cell thus the bacterium require a growth medium to survive. To show that the gene of interest has been taken in, it is important that the characteristics of the reporter gene isn't expressed beforehand, in other words, in the case of this paper, the reporter gene can only be expressed under certain conditions after plating and incubation.  The following table shows what characteristics are expected from each reporter gene:

One of the most typical uses of reporter genes is to learn about how a gene is regulated, typically a reporter gene may be found upstream of the transcription start site of a gene within the regulatory region thus anything that affects the expression of the gene should affect the reporter gene as well. In this way, reporter genes offer an advantage as we don't have to separate the regulatory regions into different sections in order to study them.

In order to determine if this recombination of DNA has worked, electrophoresis is used, this involves digesting the DNA with an enzyme that has a cutting site within the plasmid/bacterium then placing the digested samples into the wells of a polymer gel placed in buffer solution and subject to an electrical current of around 100volts.  The result is that the DNA breaks down into its digested fragments and travel across the gel (DNA is negatively charged so will run towards the positive end of the gel) with the smaller fragments travelling further than the larger ones. A DNA ladder of various fragment sizes is then used to calibrate the fragment sizes of the recombinant DNA. Thus from this we can tell if the required gene has been incorporated in the the plasmid.

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Aims

By using the characteristics of reporter genes as well as the characteristics of the bacteria themselves, we were to identify four bacteria known originally as A,B,C and D.  We had to incorporate the bacteria into plasmid vectors, plate and incubate, then use various methods to properly identify them.  After which to ensure that we had successfully recombined the DNA of the bacteria with the plasmid vectors, we had to selective an enzyme which we believed would digest the recombinants into smaller fragments for easier identification.

Other aims were to develop our laboratory skills, such ...

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