Explain Alpha-Complementation and Blue-White Screening To Me Like I’m 10

Alpha-complementation – it’s a big phrase, but it’s not terribly hard to understand.

The technical explanation of alpha-complementation involves combining two parts of a protein, making the complete functional protein. Alpha-complementation is used in molecular biology to screen for colonies that have the desired genetic construct.

How alpha-complementation works can get really sciencey. But rather than get too technical too quickly, let’s start with a really easy-to-understand analogy. After that, we’ll get into the science and then relate the analogy to the concepts.

Figure 1. One half of a treasure map. The X for the treasure is not on this half. Therefore, the map does not function the way it should.

That torn half will only get you so far. Without the other half, you cannot find the treasure.

So, for this map to properly function, you are going to need to combine your half with the missing half.

This is very similar to how alpha-complementation works, but rather than a map or something easily seen, researchers use a similar strategy at the cellular and protein level.

So let’s now start to dig into the science of it all.

E. coli is a model bacterium used in the lab for molecular biology. And it has an enzyme called beta-galactosidase that helps E. coli break down sugars like lactose.

Interestingly, the functional beta-galactosidase enzyme, capable of breaking down lactose and its analogs like X-gal, is formed by the tetramerization of four identical monomers of beta-galactosidase polypeptides.

Tetramerization – it’s kind of a mouthful. But all it means is that the four monomers (small molecules that can bond together) bond with each other to form a tetramer (a larger molecule made up of four small molecules, or monomers).

Figure 2 represents four monomers tetramerizing or forming a beta-galactosidase tetramer.

Figure 2. Illustration representing the identical monomers or beta-galactosidase polypeptides as well as the complete tetramer.

Within each beta-galactosidase enzyme, there are sequences of amino acids. When amino acid residues 11-41 are removed from the amino-terminus, the monomers cannot form the tetramer (tetramerize).

To get technical, the peptide missing these residues is called the omega peptide (ω).

Without the monomers being able to tetramerize, the complete enzyme cannot form, and therefore E. coli cannot break down x-gal.

Figure 3. Illustration of beta-galactosidase identical monomers missing amino acid residues 11-41 (ω-peptides). The result is no tetramerization, and therefore no complete beta-galactosidase enzyme is formed.

Beta-galactosidase is a functional enzyme when it is composed of the joined four identical beta-galactosidase polypeptides. This is what is called a homotetramer (same/four).

When working with this concept, researchers manipulate the sequences of these peptides to form an alpha (α) peptide and an omega (ω) peptide.

The ω-peptide is the beta-galactosidase polypeptide, but as discussed, it is missing the amino acid residues 11-41 – similar to having part of a treasure map, but not the whole part.

Scientists call this specific modification the lacZΔM15 mutation. Because of this mutation, the ω-peptides cannot form a homotetramer, which means it is not functional (figure 3).

This mutation is present in the genomes of E. coli cloning strains.

Meanwhile, there is another side of this whole equation – that missing piece that would allow the monomer to form a homotetramer. This is the α-peptide, a small sequence with amino acid residues 1-59, which complements the missing amino acids in the ω-peptide – in other words, our missing piece of the map.

Figure 4. Illustrates alpha-complementation with the ω-peptide and the α -peptide.

Once fully formed, the complete beta-galactosidase enzyme can break down x-gal.

Figure 5 below visually summarizes the whole process. In panel A, you’ll see the identical monomers represented. When they come together, forming the homotetramer, the result is a functional beta-galactosidase enzyme that can break down x-gal.

In panel B, we’re just looking at the polypeptides with the missing amino acid residues (11 – 41). Because of this missing part, beta-galactosidase polypeptides cannot tetramerize, and no enzymatic activity happens.

Finally, in panel C, we have the ω-peptide with the missing amino acid residues and the α-peptide with amino acid residues 1-59. The result is alpha-complementation – like putting our treasure map halves together, leading to functional monomers that can tetramerize to form a functional beta-galactosidase enzyme.

Figure 5. Summarizes beta-galactosidase monomers, the ω-peptide and the α-peptide along with how they interact. Panel A represents functional monomers that form a homotetramer. Panel B represents the ω-peptide missing amino acid residues 11-41. Panel C represents the ω-peptide and it’s α-peptide complement.

Now we can take a look at this process within a host cell (usually E. coli cloning strains) and a plasmid that works as a vector carrying genetic material into the host cell.

Figure 6 shows the sequence encoding the α-peptide (pink) and the sequence encoding the ω-peptide. The sequence encoding the α-peptide is found within the plasmid while the sequence encoding the ω-peptide is within the E. coli genome.

Once the plasmid containing the α-peptide is within the E. coli cell, alpha-complementation can take place.

Figure 6. The lacZα sequence encoding the α-peptide is shown in the plasmid. The lacZΔM15 sequence encoding the ω-peptide is within the E. coli genome. E. coli transformation leads to α-peptide and ω-peptide production and alpha-complementation.

The reason it is so important for scientists to use alpha-complementation in certain scenarios is that it lets them examine whether certain desired or undesired genetic changes have occurred in bacteria during the gene cloning process.

When they attempt to introduce the alpha and omega peptides back together within the E. coli cell, and they fit, then the E. coli cell will have functioning beta-galactosidase enzymes that allow it to break down x-gal.

Going back to our treasure map, imagine you have a torn half, and you’re visiting a library full of old maps – many are torn. Your goal is to find your other half, and you’ll know you have it if the two pieces you attempt to fit together match up. Once you have matched up your map to its other half, the functioning map can lead you to the treasure.

Figure 7. Combining both halves of a treasure map means the map now functions as it should, guiding adventurers to the treasure (X).

The classic application using alpha-complementation is blue-white screening.

Blue-white screening and alpha-complementation

To understand how alpha-complementation works in the technique called blue-white screening, let’s look at another analogy.

Imagine you and a friend are putting together a stackable bookshelf – you know, one of those bookshelves that comes from a regular retail store.

Conveniently, the bookshelf comes in two sections, a cabinet base and the actual shelving section that gets stacked and secured on top of the cabinet base.

Figure 8. Displays the two units of the bookshelf. When the cabinet and shelf are combined, they form the complete bookshelf.

To make the job go faster, you decide to work on the base while your friend builds the shelving section.

Fortunately, the manufacturers anticipated teamwork and crafted their instruction manual into two dividable sections – the instructions for the cabinet section and instructions for the shelving section.

You have the first section of the instructions (cabinet), and your friend has the second section (shelves).

When you each build your sections, you stack them together making the whole completed bookshelf.

Figure 9. Two people build each section of the bookshelf using the corresponding parts of the instructions. Once they put together the two bookshelf sections, it forms the full unit shown in the green rectangle.

Now, let’s reimagine this same scenario with a third friend. Your third friend is a bit of a prankster and decides to have a little fun.

When he learns that you have one of the divided parts of the instructions, the prankster decides to craft a few extra instructions of his own.

Figure 10. Prankster inserting his instructions (pink) into the cabinet instructions.

He inserts his instructions into your section of the manual. And when you follow the manufacturer’s instructions along with the instructions the prankster inserted, the base you attempt to build won’t function properly. In fact, what you build won’t really end up being a base at all.

So, when you and your friend try to combine the bookshelf base with the shelving unit, it won’t work.

Figure 11. Based on the inserted (pink) lines of instructions, a nonfunctioning cabinet is built. The shelving section cannot be stacked on the nonfunctioning cabinet (shown in the pink box).

This is conceptually similar to how researchers use the science behind blue-white screening and alpha-complementation to confirm whether the plasmid vector actually took up the genes they inserted or not. Or, another way to think of it is that it lets scientists know if the extra instructions they wanted to add actually did get added.

See, in alpha-complementation and blue-white screening, researchers use a plasmid to carry genetic information into an E. coli cell. But they have to make sure the genetic information they want is actually inserted into the plasmid. And blue-white screening will visually allow them to see this. We’ll look at the visual part of this story a little further on in this article.

Right now, to make it clearer, let’s map the players of blue-white screening to the players of this bookshelf example.

You are the vector. Your friend represents the host cell that the vector/plasmid is transformed into. And the prankster is the researcher inserting their own set of instructions.

Furthermore, the two sections of the instructions for the bookcase represent the LacZ genes to build the α-peptide and the ω-peptide. And the prankster’s instructions are like the sequence a researcher is adding to the vector.

Table 1. Analogy and molecular representation lookup.

Now let’s talk about the visual part. When doing blue-white screening, in order to visually distinguish whether the vector has the desired sequence or not, the researchers added their target gene into the vector in such a way that it disrupts the genetic code to form the working α -peptide.

Figure 12. Gene of interest (purple outline) is inserted into a section of the plasmid and the insertion breaks up the lacZα gene that encodes the α -peptide. Because the lacZα gene is disrupted, the α -peptide cannot be produced

This is similar to the prankster who added instructions into your manual. The bookshelf’s cabinet that you produced based on the prankster’s work won’t be fully functional. Just like the α-peptide with the target gene ligated into the vector.

Based on the principles of blue-white screening, blue colonies form on a plate when a functional ω-peptide combines with the α-peptide, allowing tetramerization. This is because of a blue color change that occurs when the functioning beta-galactosidase in E. coli breaks down x-gal.

Figure 13. Both the α-peptide and ω-peptide combine, allowing peptides to form a tetramer that leads to beta-galactosidase. E. coli colonies with beta-galactosidase are blue due to a color change that occurs when the enzyme breaks down x-gal.

If the gene for the α-peptide is disrupted by the insertion of a researcher’s target gene, the α-peptide isn’t even produced, and α-complementation doesn’t happen. This leads to white colonies growing on the plate since there is NO functional beta-galactosidase enzyme breaking down x-gal that would lead to a blue color change.

Figure 14. The plasmid with the target gene inserted disrupts the lacZα sequence. There is no α-peptide and no alpha-complementation that occurs. E. coli colonies with the recombinant plasmid appear white.

Here is where we put this all together. Remember that researchers are using blue-white screening to confirm if their plasmid actually took up the inserted gene or not.

And we now know that when it comes to blue-white screening, we can visually confirm if the gene was inserted because the inserted gene will disrupt the sequence that produces the α-peptide. This leads to no alpha-complementation, and results in white colonies on a plate.

When put in practice, some plasmids will successfully take up the gene while others won’t. And the results on the petri dish will have a mix of blue colonies and white colonies, which you can see in figure 15.

To move forward with the experiment, a researcher will select the white colonies since those colonies have the gene that they inserted into the plasmid.

Figure 15. Summarizes blue-white screening and colony selection. Colonies with the recombinant plasmid grow white on the petri dish. Colonies that do not have the recombinant plasmid with the desired gene inserted will appear blue.

The only difference here between the bookshelf analogy and what actually happens is that in the analogy, we still suggest that the bookshelf base is produced but doesn’t work right. In reality, the disruption in the genes for the α-peptide would mean that the α-peptide isn’t even produced.

Either way, complementation does not occur. For the researcher, they know they were successful by finding bacterial colonies with the gene that they inserted into their vector.

So that’s it. That’s the overall principle behind alpha-complementation and how it is used in blue-white screening, and thankfully, it’s not all that overwhelming in the end. We have a lot more resources on this topic. For instance, you can learn about blue-white screening within differential media, or how expression strains of E. coli are engineered for blue-white screening are engineered for blue-white screening.