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For more information, visit our partner 3-D Molecular Designs. The GFP expressed from the pGLO plasmid illustrates the central doctrine of biology, from the transformation of DNA to the expression of a protein to the visualization of a trait.
The bacterial proteome contains thousands of proteins, but only the cloned GFP glows! In its native environment, GFP fluoresces in the deep sea jellyfish, Aequorea victoria. Incredibly, GFP retains its fluorescent properties when cloned and expressed in E. These extensions link two of the most commonly used techniques in biotechnology labs: transformation and protein purification. Purification of a protein depends on a its chemical or physical properties, such as molecular weight, electrical charge, or solubility.
GFP can be separated from others by its size using electrophoresis, and it is extremely hydrophobic, which enables its purification using hydrophobic interaction chromatography HIC.
When placed in a buffer containing a high concentration of salt, the HIC matrix selectively binds hydrophobic GFP molecules while allowing the bacterial proteins to pass right through the column. Then, simply lowering the salt concentration of the buffer causes GFP to elute from the column in a purer form. Students can explore these separation techniques by growing transformed bacteria in liquid culture to grow overnight, then lysing the cells to release their contents.
The unique fluorescent property of GFP allows real-time monitoring of extraction and purification, modeling key processes used in biotechnology to produce and purify designer proteins with commercial or research value. Use these short, instructional videos to enrich lessons about bacteria, bacterial transformation, and the green fluorescent protein GFP.
Investigate the functional elements of pGLO bacterial transformation, including heat shock, antibiotic selection, promoters, and satellite colony formation. You can create and edit multiple shopping carts Edit mode — allows you to edit or modify an existing requisition prior to submitting.
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My Account. Browse Catalog. Life Science Research Back. Life Science Research Explore all. Bio-Rad Products Explore all. Sat I is different from the others. First, its restriction site is only five nucleotides long. Also, in this restriction site, "N" in the middle means that any nucleotide will work; as long as Sat I finds the other nucleotides in the sequence, it will cut the DNA.
As mentioned earlier, you're going to cut your plasmids so you can tell them apart. Plasmids normally exist as circular, double-stranded DNA molecules in bacterial cells. In addition to the double helix secondary structure found in almost all DNA, these circular DNA molecules can wind around themselves to form tightly coiled, compact supercoiled conformations.
Thus, this type of DNA has tertiary structure. When you compare your supercoiled plasmid DNA to a linear molecular weight marker as you did on the total nucleic acid gel , you don't get a true indication of the size of the plasmid. This can make it difficult to know whether you are seeing the plasmid that you expect.
Keep in mind that there are usually multiple copies of the plasmid in a single cell. Even if most of the plasmid DNA is supercoiled, a small percentage of it might be in the linear if both strands got broken or open circle if one DNA strand got broken, allowing the supercoiling to untwist.
Thus, you could potentially have several bands of plasmid DNA on a gel, even if all the plasmid molecules are the same length and same molecular mass. Cutting circular DNA with a restriction enzyme that cuts at only one site will linearize the DNA convert it from circular, supercoiled form to linear form.
This allows for more precise determination of the size of the DNA fragments. In this lab experiment, you will start with two different plasmids that are close to the same size pGLO is base pairs long, and pARO is bp.
When they are supercoiled, it might be difficult to tell them apart on a gel. Each of these plasmids has one restriction site for Eco RI, so this enzyme will linearize them, making it easier to tell them apart. You can get more information by using restriction enzymes that cut more than once.
That's a good way to tell these plasmids apart. It's a bacteriophage, meaning it's a virus that infects bacteria. DNA maps are simplified representations of nucleotide sequences, showing features of particular interest such as genes. Restriction maps are DNA maps that show restriction sites, the locations where specific restriction enzymes will cut the DNA.
Here is a map of pGLO:. I made this map with Benchling , the online software package that I usually use for analyzing nucleotide sequences. This map will give you a general idea of what size DNA fragments to expect if you cut pGLO with each of these enzymes:.
Now suppose you want to cut the plasmid with more than one enzyme at a time. Each enzyme will act on its own restriction sites, so the different enzymes won't affect each other. From the map above, you can see that if you cut with both EcoRI and HindIII, you will cut out a tiny piece between the EcoRI and HindIII sites; you probably wouldn't be able to see this small fragment on a gel, so it's not worthwhile to perform this digest.
Using this slightly more detailed map courtesy of Snapgene , you should be able to predict the exact sizes of the DNA fragments you will get:. This map shows the exact locations of the restriction sites. Each nucleotide is assigned a number, based on an arbitrarily chosen starting point. If PvuI cuts at nucleotide and EcoRI cuts at nucleotide , and the entire circular plasmid is bp long, you can probably figure out the sizes of the fragments you will get.
Restriction Analyzer from molbiotools. For a precise electronic restriction digest, you need to enter the nucleotide sequence of the DNA you're going to cut. Find the pGLO sequence the original sequence from Bio-Rad on the Sequence Data page keep that page open in another tab; you'll use it again.
Copy the whole sequence, including the numbers, and paste it into the "sequence info" box in the Restriction Analyzer the software will ignore the numbers and spaces, and only look at the nucleotide sequence.
Note that the sequence is shown for only one strand of DNA, but the software is smart enough to recognize that the actual DNA is double-stranded. Click on Virtual Digest. Virtual digest will take you to a page showing the complete nucleotide sequences of the DNA fragments produced in the digest which you probably don't care about and also the length of each fragment which you will be able to verify on your gel.
Those fragment lengths are your expected results for this experiment. Alternative approach: You could also use Benchling instead of RestrictionMapper. Thus, you need to save some transformed colonies. Depending on your plate results, you might want to restreak some bacteria from one of your transformation plates onto a new plate.
Check with your instructor to see whether you should do this today. This will give you additional fresh colonies to use in later labs. Use the technique from starting bacterial cultures. For this lab, you should restreak on a plate with ampicillin to ensure that only cells containing the pGLO plasmid will grow.
Arabinose is optional in this plate, but might be helpful; discuss this with the instructor. Once you restreak a plate, put it in the incubator so it can grow. Do not seal this new plate with tape. In addition to the newly restreaked plate described above, you should save some of your transformation plates to make sure you have colonies for later use.
If your results are good, save plate 1 , which has fluorescent pGLO-transformed colonies. If you don't have good colonies on that plate, you may be able to use another plate that has colonies containing pGLO; this could be any plate with ampicillin.
Also save plate 6. This plate may contain colonies with different types of pGLO, producing green, blue, or non-fluorescent colonies. Tu, American Society for Microbiology. Excellent description of the classic protocol. How to perform a bacterial transformation from Bio-Rad Explorer. Good, clear video that is specifically about the pGLO experiment. We do some slight variations on this in 6B, so don't follow the video instructions exactly.
Competent Cell Transformation from Invitrogen. How to Transform E.
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