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September 19, 2020, 10:13:50 pm

Author Topic: Recombinant Bacteria Selection  (Read 133 times)  Share 

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marchy7

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Recombinant Bacteria Selection
« on: August 12, 2020, 04:36:19 pm »
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Hi,

In class I was taught that selecting transformed bacteria involved using two separate antibiotics;

1. Pores are created in the bacteria's plasma membrane using heat shock or electroporation, allowing the bacteria to take in the plasmids.
2. The bacteria are placed on a dish containing antibiotics; the bacteria that took up the plasmids (with an antibiotic resistant gene) will survive and grow. The bacteria without the plasmid are killed by the ampicillin.
3. The surviving bacteria are exposed to another dish with a different antibiotic. The bacteria that do not survive contain the recombinant plasmid with the disrupted antibiotic (tetracycline) gene.
4.  The culture of the bacteria with the recombinant plasmid are grown on another dish and are induced to express the gene of interest to produce the corresponding protein.

However, other sources say that there is only one exposure to antibiotics; an antibiotic resistant gene is inserted into the plasmid, resulting in the survival of only the transformed bacteria.

I was just wondering which one was correct because all the other notes said that it was one exposure but my teachers said that was wrong.  :-\ :-\


Mod edit: You posted the exact same post twice so I deleted one of them - Bri
« Last Edit: August 12, 2020, 04:44:10 pm by Bri MT »

-Lilac-

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Re: Recombinant Bacteria Selection
« Reply #1 on: August 12, 2020, 05:37:56 pm »
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Hi!

When I was in VCE I was just taught that following the transformation of bacteria with a plasmid they are then grown on antibiotic-containing media to select for the transformed bacteria. I am not sure what the study design states now though (maybe someone else has some insight).

However, what your teacher has described is not incorrect.

Before you transform your bacteria you have to clone your gene of interest into the plasmid. When you do this, there are two possible outcomes as you mentioned. A recombinant plasmid which has taken up the gene or an 'empty' plasmid (that still has the ampR gene). Thus, you not only want to select for bacteria that have taken up a plasmid but have taken up the recombinant plasmid.

How I usually do this is blue/white selection or colony PCR (don't worry about those terms though). But I did some research and it turns out you can replicate the colonies from the amp plate onto a plate containing tetracycline. You then compare the two plates and the colonies that are on the amp plate but missing from the tetracycline plate are the bacteria with the recombinant plasmids.

angrybiscuit

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Re: Recombinant Bacteria Selection
« Reply #2 on: August 12, 2020, 10:18:22 pm »
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Hi there!
This is from my understanding.

There are two ways in detecting transformed bacterium:
1. Via a lacz gene (or another equivalent gene)
2. Via another antibiotic (which is what your teacher has said)

Essentially you have one for antibiotic resistance, and the other a "marker" gene. This "marker" gene has to be disrupted because the foreign, desired gene is inserted meaning it cannot be transcribed.

Thus there are also two ways in creating recombinant plasmids:
1. You can have a plasmid with 1 gene for antibiotic resistance and the other a lacz gene.
2. You can have a plasmid with 2 different genes for antibiotic resistance

The lacz gene is translated into an enzyme that catalyses colourless X-gal into product that is blue. (This is very important!)

Then as you know, when creating transformed bacteria we get three possible outcomes.



With two antibiotic-resistant genes
[Ampicillin gene and tetracycline gene (marker gene)]
1. No plasmid at all.
> These will be killed in a dish with antibiotics (eg, ampicillin) because they do not have any genes for antibiotic resistance

2. Plasmid but without the foreign gene. Therefore "marker" gene is not disrupted.
> They will not be killed by ampicillin because they have the antibiotic resistance gene. They will also not be killed by tetracycline as this gene is not disrupted. This bacterium is not transformed. 

3. Plasmid but with foreign gene. Therefore "marker" gene is disrupted.
> They will not be killed by ampicillin because they have the antibiotic resistance gene. They will be killed by tetracycline as this gene is disrupted. This bacterium is transformed. 

With lacz gene and antibiotic-resistant gene
[Ampicillin gene and lacZ gene]
1. No plasmid at all.
> These will be killed in a dish with antibiotics (eg, ampicillin) because they do not have any genes for antibiotic resistance

2. Plasmid but without the foreign gene. Therefore "marker" gene is not disrupted.
> They will not be killed by ampicillin because they have the antibiotic resistance gene. LacZ gene is translated as the gene is not disrupted. X gal is formed into a blue product. So these bacteria colonies will be blue.

3. Plasmid but with foreign gene. Therefore "marker" gene is disrupted.
> They will not be killed by ampicillin because they have the antibiotic resistance gene. LacZ gene is not translated as the gene is disrupted. X gal remains colourless. So these bacteria colonies will be colourless.

Therefore, the colourless colonies are those with the transformed plasmid.

TLDR; there are multiple methods in finding out whether or not the bacterium has the recombinant plasmid. You can have one or two antibiotics exposure depending on the genes the plasmid has. 
somewhere, something incredible is waiting to be known.
carl sagan