Cloning w/ Plasmid DNA: AmpR Gene Overlap Issue

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In summary, the problem involves a plasmid DNA with a restriction site overlapping with the AmpR gene. This may cause inactivation of Ampicillin resistance when the restriction enzyme cuts the site. However, through the process of replica plating, bacteria that have taken up the plasmid can be distinguished from those that haven't by incorporating a gene of interest within the antibiotic resistance gene. This allows for the identification of bacteria with the desired gene of interest.
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plexus0208
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Homework Statement


I have a problem that involves a plasmid DNA. The problem gives a plasmid diagram with the restriction sites and specifically tells me which restriction site the gene of interest would go. However, the restriction site is shown to be overlapping with the AmpR gene. Wouldn't that mean the Ampicillin resistance is deactivated when the restriction enzyme cuts the site? I don't get how the gene is supposed to be amplified in bacteria if the plasmid doesn't have AmpR...

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Biology - none

The Attempt at a Solution


See above
 
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  • #2
Hello!
I think the process you are reffering to is replica plating. It allows you to keep the colonies that you have produced in the experiment, whilst checking for certain genotypes. Some bacteria may take up the plasmid but not the recombinant form containing a gene of interest, by placing it within an antibiotic resistance gene, it inactivates that gene. There will be other resistant genes also present on the plasmid, and yopu can check the colonies against a number of antibiotics; this two step process allows you to distinguish between bacteria that have taken up the plasmid from those that haven't, determined by a functioning resistance gene on the plasmid that is not disrupted by restriction endonucleases; and then the colonies formed from the bacteria of interest (those with a plasmid that have incorporated a gene of interest) can then be distingiushed from those that have taken up a plasmid but do not contain the gene of interest, as those that have not taken up the gene of interest will be resistant to the second type of antibiotic, but the those of interest will not be resistant. Remember, replica plating does not destroy the colony of bacteria!
I think this is right, I hope it helps.
 
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1. What is the purpose of cloning with plasmid DNA and the AmpR gene?

The purpose of cloning with plasmid DNA and the AmpR gene is to create multiple copies of a specific gene or DNA sequence. This allows for amplification and purification of the desired DNA, making it easier to study and manipulate.

2. What is the main issue with cloning using plasmid DNA and the AmpR gene?

The main issue with cloning using plasmid DNA and the AmpR gene is the potential for overlapping of the gene sequence. This can occur if the gene sequence being cloned is similar to the AmpR gene, leading to incorrect amplification and potentially altering the function of the desired gene.

3. How can the issue of AmpR gene overlap be avoided during cloning?

The issue of AmpR gene overlap can be avoided by carefully selecting the plasmid and gene sequences being used for cloning. It is important to choose plasmids with unique AmpR gene sequences and to carefully design primers for the gene being cloned to minimize any potential overlap.

4. What are the potential consequences of AmpR gene overlap during cloning?

The consequences of AmpR gene overlap during cloning can include the incorrect amplification of the desired gene, resulting in the production of incorrect proteins or altered gene function. It can also lead to difficulties in the purification and study of the desired gene.

5. Are there any alternative methods for cloning that can avoid the issue of AmpR gene overlap?

Yes, there are alternative methods for cloning that can avoid the issue of AmpR gene overlap. These include using alternative selection markers in plasmids, such as KanR or TetR, or using PCR-based cloning techniques which do not rely on the AmpR gene for selection.

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