Overview

What is SPACE-P?

- Combining interesting technologies to make way for an innovative idea is what SPACE-P is all about! We'll be exploring the space within bacteria to carry-out our madness!

SPACE-P stands for Structural Phage Assisted Continuous Evolution of Proteins.

Okay, why do we need SPACE-P?

- In SPACE-P we propose an alternative way of identifying potential antibody binding partners. Our goal is to speed up screening for peptides or identified target proteins of diseases capable of interacting with antibodies.

But how does SPACE-P translate to your goal?

- We used one of the smallest antigen binding molecules currently known, the ‘affibody’, instead of monoclonal antibody. We try to evolve an affibody (ZHER2) molecule to generate a better fit to the protein of interest in our case HER2 (Human epidermal growth factor receptor) to present a new way to identify potential drug epitopes.

You can read more about affibodies here.

So how do you plan to do it?

- We combine the 3 different techniques of "Phage Display", "BACterial Two Hybrid (BACTH) system", and "Phage assisted continuous Evolution" to evolve the affibody that fits the target protein. We use a lock and key model to evolve the affibody protein.

How does this process work?

- We try to evolve the antibody which is encoded in the M13's phage genome. The target protein is encoded in the E. coli genome. Now, naturally the M13 would try to infect the E. coli to replicate. Here's the catch, we use the BACTH system as a lock and key model on the E. coli strain and the M13 phage DNA.

The lock in question is a 3-step lock system that is designed in such a way that:

1. The 2 domains of the enzyme adenylate kinase are split, one subsequence is bound to the affibody and is located in the M13 phage genome, and the other is in an accessory plasmid in the E. coli system and connected to the target protein. These two domains have to interact to be able to produce cAMP.
2. The protein P3 that is essential for phage infectivity is deleted in the M13 bateriophage.
3. The bacterial system we use is a cya- strain, which means it's defective for the enzyme adenylate cyclase.

That seems to be a pretty complicated lock system, how is this lock opened?

• Stage 1: When the M13 phage attacks the E. coli and the genome replicates so that the target protein, affibody as well as the BACTH domains are produced (with the aid of an external F plasmid in the E. coli).
• Stage 2: When the affibody and target protein come together, they also bring the adenylate cyclase domains close together. This allows for dimerization of the kinase domains as well as phosphorylation, which leads to the production of cAMP.
• Stage 3: This cAMP now has the ability to bind to the lac promoter on the E. coli genome, which then transcribes the P3 gene producing protein P3 necessary for the phages infectivity, tagged along with a β-galactosidase protein (which is our reporter). The M13 then use the P3 proteins.

When we consider that our reporter gives a read out then we can safely assume that the adenylate kinase domains interacted and that means that there was an interaction between the affibody and the target protein.

How does the M13 phage replicate and infect other E. coli cells?

- The M13 genome does not code for the P3 protein (affibody gene is inserted in place of P3); so it cannot infect E. coli cells initially.

The only way for the M13 phage to infect other cells is to activate the lacZ promoter on the E. coli genome which in turn produces the P3 protein.

Nevertheless, for the initial entry or first attack a small population of E. coli cells are facilitated to have F+ plasmid. For the later processes, they are removed and only E. coli without the F+ plasmid is fed into the reaction chamber.

But, how do you force the M13 phage to produce the P3?

- Well, here comes the most interesting part! If you subject the phage to an evolutionary stress it will try to evolve and break the lock system designed.

So, we tried to invoke evolutionary stress by removing or washing out the E. coli cells, which the initial phage culture has to attack within a span of minutes within the reaction chamber. If the attack on the E. coli isn't successful, the phages are also washed out of the lagoon.

In addition to this a mutagenesis plasmid is present in the E. coli genome used which renders the proof reading mechanism of its DNA polymerase obsolete. This introduces mutation during replication of DNA, meaning random mutation are incorporated in the resulting phage genome containing the affibody while E. coli are washed out. Therefore, this process is called phage assisted continuous evolution (PACE)!

You can read about this concept, in the Background section of our wiki. This is also the starting point of our ingenious idea!

Woah, this looks like a cool project! Best of luck for your competition!

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