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In this article, we will discuss why a promoter may have to be transferred into an organism as well as the desired gene. Moreover, we will also discuss how gene expression may be confirmed by the use of marker genes coding for fluorescent products. But before proceeding to discuss the promoters and markers, first, let us recall what is genetic engineering.
Genetic Engineering – An Overview
We can define genetic engineering as:
The technique by which scientists artificially manipulate, modify and recombine DNA or other nucleic acid molecules to alter an organism or population of organisms is referred to as genetic engineering.
The term genetic engineering is usually employed to point out the methods of recombinant DNA technology. These methods came out as a result of primary research in microbial genetics. The techniques used in genetic engineering have resulted in the production of life-saving medical products which include human growth hormone, human insulin, and hepatitis B vaccine. Moreover, they have also led to the development of GMOs, i.e. genetically modified organisms. Examples of such organisms include disease-resistant crops.
Steps Involved in Genetic Engineering
The following steps should be taken to genetically engineer an organism:
- Identify the desired gene
- Isolate the desired gene through the following steps:
- Using the enzymes restriction endonucleases, cut from the chromosome
- Create a single strand of complementary DNA (cDNA) from mRNA by employing a reverse transcriptase
- Use nucleotides to create the artificial gene
- Multiply the gene using PCR which stands for the polymerase chain reaction
- Use vectors to transmit it into an organism. Examples of vectors include liposomes, viruses, and plasmids
- Identify the cells with the new gene by utilizing the marker which is cloned afterward
Tools Required to Modify an Organism
In order to modify an organism, genetic engineers use the things listed below:
- Enzymes, for instance, ligase, reverse transcriptase, and restriction endonucleases
- Vectors that are employed to transfer the genes into the cell. For instance, viruses, plasmids, and liposomes
- Markers which refer to the genes that code for detectable substances that are also traceable
In the next section of the article, we will discuss why a promoter may have to be transferred into an organism as well as the desired gene.
Genetic Engineering: Promoter
The promoter (a length of non-coding DNA that performs a particular function) refers to the section of DNA that determines the type of gene that needs to be expressed. This is because it is a site where RNA polymerase binds to start transcription
- The promoter also guarantees that RNA polymerase can comprehend which strand is the DNA template strand. RNA polymerase comprehends the template strand because there is a transcription start point in the promoter which is the place of enzyme binding. The transcription start point refers to the first nucleotide of the gene to be transcribed
- Hence, a promoter is employed for regulating the gene expression because only in its presence the transcription and subsequent expression of the gene can take place
- If genetic engineers need to guarantee the expression of the desired gene while modifying the plasmid, then they need to add a suitable promoter
- With eukaryotic cells, bacteria entail several different genes coding for several different proteins. Although all of the genes are not switched on at once. If the growing conditions demand a certain protein, only then the bacteria will express genes to make proteins. For instance, coli bacteria make β-galactosidase enzymes only when their growing medium entails lactose but does not contain glucose
- Scientists used this information to produce insulin for diabetic patients. To do so, they added the insulin gene along with the β-galactosidase gene to share the promoter. When the bacteria wanted to metabolize lactose, the promoter switched on the gene. Hence, when the scientists started growing bacteria in a medium where lactose was present, but glucose was absent, the bacteria produced human insulin and β-galactosidase
Important point: While describing how a plasmid is genetically engineered, make sure to mention the promoter because it causes the expression of the desired gene.
In the next section of the article, we will discuss how gene expression may be confirmed by the use of marker genes coding for fluorescent products.
Genetic Engineering: Marker Genes
We can define a marker gene as:
A gene that is transferred with the desired gene to help scientists identify which cells have been modified successfully and now have recombinant DNA
- Once, antibiotic-resistant genes were commonly employed as marker genes. The bacteria were genetically modified by the scientists so that the plasmid had the desired gene along with a particular antibiotic-resistant gene and promoter.
- After that, bacteria were grown on agar plates embedded with that antibiotic
- Because only those bacteria grew that contained recombinant plasmids, hence scientists were able to identify them easily
Concerns of scientists
By utilizing the antibiotic-resistant gene as marker genes, the scientists have the following concerns:
- The risk of accidental transfer of antibiotic-resistant genes to other bacteria which include pathogenic strains creating pathogenic antibiotic-resistant bacteria is present
- The spread of resistance to other bacteria can make the antibiotics less effective
Spread of Antibiotic-resistant Genes
The antibiotic-resistant genes can spread because of conjugation which refers to the transfer of genetic material from one bacterium to another. It can also take place due to transduction which refers to the transfer of genetic material from one bacterium to another through a virus. Hence, genes that express fluorescent proteins are now usually employed as markers.
Fluorescent Genes
The presence of a green fluorescent protein (GFP) makes them fluorescent. The GFP along with the desired gene are associated with the particular promoter and after the activation of this promoter and expression of the protein, the recombinant bacteria are identified as they glow green when exposed to ultraviolet light.
Why fluorescent genes are preferred as markers?
It is preferred to use the fluorescent genes as markers because of the following reasons:
- Ease of identification (as they glow in the presence of ultraviolet light)
- They are more economical because they do not require growing the bacteria on plates of agar infused with antibiotics
- Zero risk of antibiotic resistance being transferred to another bacteria
- Such antibiotics are present which have lost their efficacy and hence they are unable to stop the growth of any bacteria
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