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Bacterial transformation & selection
Transfer of plasmid DNA into bacteria. How bacteria are selected. Protein production and purification.
Key points:
- Bacteria can take up foreign DNA in a process called transformation.
- Transformation is a key step in DNA cloning. It occurs after restriction digest and ligation and transfers newly made plasmids to bacteria.
- After transformation, bacteria are selected on antibiotic plates. Bacteria with a plasmid are antibiotic-resistant, and each one will form a colony.
- Colonies with the right plasmid can be grown to make large cultures of identical bacteria, which are used to produce plasmid or make protein.
The big picture: DNA cloning
Transformation and selection of bacteria are key steps in DNA cloning. DNA cloning is the process of making many copies of a specific piece of DNA, such as a gene. The copies are often made in bacteria.
In a typical cloning experiment, researchers first insert a piece of DNA, such as a gene, into a circular piece of DNA called a plasmid. This step uses restriction enzymes and DNA ligase and is called a ligation.
After a ligation, the next step is to transfer the DNA into bacteria in a process called transformation. Then, we can use antibiotic selection and DNA analysis methods to identify bacteria that contain the plasmid we’re looking for.
Steps of bacterial transformation and selection
Here is a typical procedure for transforming and selecting bacteria:
- Specially prepared bacteria are mixed with DNA (e.g., from a ligation).
- The bacteria are given a heat shock, which causes some of them to take up a plasmid.
- Plasmids used in cloning contain an antibiotic resistance gene. Thus, all of the bacteria are placed on an antibiotic plate to select for ones that took up a plasmid.
- Bacteria without a plasmid die. Each bacterium with a plasmid gives rise to a cluster of identical, plasmid-containing bacteria called a colony.
- Several colonies are checked to identify one with the right plasmid (e.g., by PCR or restriction digest).
- A colony containing the right plasmid is grown in bulk and used for plasmid or protein production.
Why do we need to check colonies?
The bacteria that make colonies should all contain a plasmid (which provides antibiotic resistance). However, it’s not necessarily the case that all of the plasmid-containing colonies will have the same plasmid.
How does that work? When we cut and paste DNA, it's often possible for side products to form, in addition to the plasmid we intend to build. For instance, when we try to insert a gene into a plasmid using a particular restriction enzyme, we may get some cases where the plasmid closes back up (without taking in the gene), and other cases where the gene goes in backwards.
Why does it matter if a gene goes into a plasmid backwards? In some cases, it doesn't. However, if we want to express the gene in bacteria to make a protein, the gene must point in the right direction relative to the promoter, or control sequence that drives gene expression. If the gene were backwards, the wrong strand of DNA would be transcribed and no protein would be made.
Because of these possibilities, it's important to collect plasmid DNA from each colony and check to see if it matches the plasmid we were trying to build. Restriction digests, PCR, and DNA sequencing are commonly used to analyze plasmid DNA from bacterial colonies.
Protein production in bacteria
Suppose that we identify a colony with a "good" plasmid. What happens next? What's the point of all that transforming, selecting, and analyzing?
Possibility 1: Bacteria = plasmid factories
In some cases, bacteria are simply used as "plasmid factories," making lots of plasmid DNA. The plasmid DNA might be used in further DNA cloning steps (e.g., to build more complex plasmids) or in various types of experiments.
In some cases, plasmids are directly used for practical purposes. For instance, plasmids were used to deliver a human gene to lung tissue in a recent gene therapy clinical trial for patients with the genetic disorder cystic fibrosisstart superscript, 1, end superscript.
Possibility 2: Bacteria = protein factories
In other cases, bacteria may be used as protein factories. If a plasmid contains the right control sequences, bacteria can be induced to express the gene it contains when a chemical signal is added. Expression of the gene leads to production of mRNA, which is translated into protein. The bacteria can then be lysed (split open) to release the protein.
Bacteria contain many proteins and macromolecules. Because of this, the newly made protein needs to be purified (separated from the other proteins and macromolecules) before it can be used. There are a variety of different techniques used for protein purification.
In one technique called affinity chromatography, a mixture of molecules extracted from the lysed bacteria is poured through a column, or a cylinder packed with beads. The beads are coated with an antibody, an immune system protein that binds specifically to a target molecule.
The antibody in the column is designed to bind to our protein of interest, and not to any other molecules in the mixture. Thus, the protein of interest is trapped in the column, while the other molecules are washed away. In the final step, the protein of interest is released from the column and collected for use.
Explore outside of Khan Academy
Do you want to learn more about bacterial transformation? Check out this simulation from LabXchange.
Do you want to learn more about the selection of transformed bacteria? Check out this simulation from LabXchange.
LabXchange is a free online science education platform created at Harvard’s Faculty of Arts and Sciences and supported by the Amgen Foundation.
Want to join the conversation?
How are the proteins bound to the antibodies, in the affinity chromatography, released?
And how are these antibodies formed or selected to match and pick up the proteins?(9 votes)
When you use the chromatography, you (always) need to use several buffers each with different salt concentrations to purify the protein. The proteins react to the presence of salt, so it would be whether the proteins would stick to the resin or not (this really depends on what protein you are using) or the proteins would unfold or not. Some of the main buffers that many labs use are:
Column Equilibration Buffer (2M) is to store all of the components in the columns so that they don't dry up.
Binding Buffer (4M) unfolds the proteins so that the hydrophobic proteins stick to the resin and the hydrophilic proteins pass through the column.
Wash Buffer (1.3M) releases the moderately hydrophobic proteins from the resin (lower salt conc.).
Elution Buffer (0.1M) releases the proteins (lowest salt concentration).
I hope this made sense..^^(9 votes)
Why cant bacterial plasmid vectors be used to transform plant cells?(2 votes)
Well, they can...but it depends what kind of bacteria and what kind of plasmid. To transform a plant cell, you'd want a plasmid vector that could be replicated in Agrobacterium tumiefaciens. Agro is a bacterium that can insert segments of DNA into the genome of a plant cell, and it's used to generate stably transformed plants. To transform a plant cell, you'd need to insert your gene into an Agro-compatible vector, between two sites in the plasmid that "tell" the bacterium which segment of DNA to transfer. Then, if you infected plant cells with the plasmid-carrying bacteria under the right conditions, you could get transformed cells with the gene of interest inserted into the DNA.
However, you couldn't use just any bacterial plasmid vector to make a transgenic plant. For instance, a plasmid that replicates in E. coli (as described in this article) could not be used to stably transform plant cells.
Hope that helps!(15 votes)
Can we use Calcium chloride in Solution to make bacteria more permeable instead of Heat Shock?(4 votes)- There are certainly different methods of DNA sample preparation, and calcium chloride is one of them! Depending on the type of bacteria you use and the analysis methods you plan on using, certain methods are better than others (and most are used in parallel). Calcium chloride is typically used with heat shock to prepare what is called "competent cells." Competent cells are cells which have been treated (typically with calcium chloride) to improve the success of transformation. DNA is negatively charged, so the calcium cations in calcium chloride bond to the negatively charged DNA, creating an overall neutral charge. This reduces electrostatic repulsion and assists with the success of the heat shock!(7 votes)
- How does transformation ensure that a bacteria will get only one plasmid?(5 votes)
- Good question.
For a typical transformation (e.g. an insert ligated into a vector) you would have something on the order of 10⁹ to 10¹⁰ DNA molecules and maybe 10⁷-10⁸ bacteria.
However, transformation is an inefficient process — you will typically get less than a thousand colonies (often many fewer).
Thus, the chance that any one bacteria would get two plasmids is extremely low (around 10⁻⁴ - 10⁻⁵).
Note also that in many situations you are only transforming one version of a plasmid, so it wouldn't matter even if you did get multiple copies of a plasmid.
Does that help?(4 votes)
- DNA lygase requires ATP but we ant providing any ATP in ligation reaction. then how it works??(3 votes)
- Correct, the DNA ligation reaction requires ATP. A typical ligation reaction involves incubating the plasmid, DNA fragment of interest and DNA ligase in a ligation buffer. We typically use a ligation buffer that contains ATP which allows the reaction to take place.(7 votes)
How can bacterial transformation can be used in the field of science? What are some real-life applications of this process?(3 votes)
Real-life application is for the needs of the Biotechnology industry and research.
Whenever you need to develop new drug treatment, to test antibiotic resistance of bacteria, to use bacteria, for genetic engineering and mainly for gene expression studies.
So you use bacteria to 'force' gene of interest into her and then grow on the medium. After selected colonies (those who have plasmid growing up on selective medium with antibiotic) you also use chromogenic colours to visualize the expression of certain proteins in those colonies.
https://edvotek.wordpress.com/2014/07/18/biotechnology-basics-bacterial-transformation/
In nature, bacterial transformation happens common, when developing antibiotic resistance and bacteria then sned their resistant even to another one (horizontal transmission).(4 votes)
- Wouldn't it be hard to find a restriction enzyme for a particular gene of interest because the desired gene must have the recognition site for the restriction enzyme on both ends?(2 votes)
- First, most vectors will have a region known as the "Multiple Cloning Site" (MCS) that can be cut with many different restriction enzymes† — this gives you more choices of enzyme and makes it more likely that you can find one that cuts near the ends of the region you wish to clone.
Second, we often don't care if we clone a small amount of extra DNA , this means that we can search over a larger area than you might expect to find appropriate restriction enzymes.
Third, we don't need to use the same enzyme for both ends. In fact, it is quite common to use two different enzymes and this allows us to do "directional cloning" — i.e. the different ends mean the insert can only be put into the plasmid in one orientation‡. This again greatly increases the number of possible restriction enzyme sites.
If the regions flanking the sequence you want to clone don't contain any useful restriction sites you can instead use primers with restriction sites added to their 5' ends and then amplify the sequence using PCR§.
This amplifies the insert you want and creates a copy of the insert DNA with whatever restriction sites you want added at the ends.
There are many more tricks that have been developed, but adding sites at the ends of primers almost always works, so that is a very good one to know!
Does that help?
†Note: There are hundreds of commercially available restriction enzymes recognizing many different sequences (many of which are palindromes, but not all).
Among these the most commonly used are six-cutters (with 6 bp recognition sites — if you make a bunch of simplifying assumptions you can calculate that these enzymes on average will cut once every 4096 bp.
‡Note: For some applications this can be very important, for example if you are using an expression vector you need the insert to transcribed in the correct direction!
§Note: Polymerase chain reaction — you can learn more about this technique here:
https://www.khanacademy.org/science/biology/biotech-dna-technology#dna-sequencing-pcr-electrophoresis(5 votes)
Wont some of the bacteria that didnt take up the recombinant plasmid have their own plasmids that have antibiotic resistant gene such as ampicillin so that even they survive and appear in the colony?(2 votes)- Good question!
It could be difficult to know if you were just using a random bacteria isolated from nature — especially since there are likely to be many thousands of different plasmids (1730 were present in a sequence database as of 2009). We could sequence all the DNA inside the bacteria, but that is still a lot of work ...
However this doesn't matter as much as you might think.
For example, assume we are using a plasmid that contains a marker (selectable gene) encoding resistance to ampicillin. All we need to know is that the bacteria were are transforming are not already resistant to ampicillin. This is easy to test — we just try growing the bacteria in the presence of ampicillin, if they don't then we can use our plasmid.
In practice microbiologists have domesticated strains of bacteria (a favorite is Escherichia coli — often abbreviated to E. coli) that have been studied for decades. In almost all cases you would be using one of these well characterized strains and so would not need to worry about whether there were unknown plasmids.(4 votes)
- How can reporter genes be used to separate bacteria who have taken up the transformed plasmid from those who have taken up the non-transformed plasmid?(3 votes)
- That's why reporter genes exist - usually, antibiotic-resistant genes are used as markers (such as Tet - resistance to Tetracycline).
IN that case, all bacteria would be inoculated onto antibiogram including Tetracycline.
If Bacteria grow, it means that colonies carry transformed plasmid. If they do not grow they do not carry transformed plasmid.
Antibiogram is used to selectively separate bacteria.(2 votes)
- My textbook says small size vectors are preferred for cloning. Why?(2 votes)
- Larger vectors are more likely to contain duplicates of the restriction sites and so are harder to work with — you typically will cut at unique restriction site(s)§ when cloning, but these are harder to find in larger vectors.
Larger vectors are also more likely to be broken by "shear" forces in a solution — e.g. when the solution is being pipetted.
Finally, the efficiency of ligation and transformation tends to decrease with larger vectors.
A typical plasmid can be up to total size of around 50 kb, but plasmids that are more than 20 kb are very difficult to work with and may require special transformation techniques.
In particular, larger vectors are more likely to
Does that help?
§Note: These are often found clustered together in a "Multiple Cloning Site" (MCS).(3 votes)
