Team:UC Chile/Disruption




Once the desired product is inside the Carboxysome, a method for the recovery of such a product becomes necessary. Since our project is a novel application for this microcompartment, not many available protocols for such purpose are available. Thus, we design a method that will allow us to purify products made or stored inside this microcompartment. This includes a process by which we could get the Carboxysome out of the bacterial cell and release its content out of it.

Bacterial Autolysis

The first step is to take the Carboxysome out of the bacteria that hold it. This will be achieved with the development of an autolysis device that produces the degradation of the inner membrane and the bacteria cell wall. This device will be composed from several BioBricks already available in the Registry of Standard Biological Parts: the “Enterobacterial phage T4 lysis device” (BBa_K112808) that is composed by the T4 Holin (BBa_K112805), which forms pores in the membrane of the bacteria; and the T4 Endolysin (BBa_K112806), which is the lysozyme that degrades the peptidoglycans presents in the bacterial cell wall. Together, this two will kill the bacteria. The device also includes an antagonist that binds to the Holin and prevents its activity unless produced by induction.

This BioBrick does not include a promoter, so we propose two options:

Add the OsmY promoter (BBa_J45992): a stationary phase promoter that will induce the lysis of the bacteria once they reach its growth phase. This works under the assumption that for that point in the growth, all the bacteria will have all their Carboxysomes at their maximum capacity. The advantage of this method is that the system will work more independently. On the other hand, there is an issue with the basal expression of this promoter which that needs to be established and compared with the expression of the antiholin promoter (BBa_J23116) to avoid lysis before the stationary phase. Although, since this is a constitutive promoter, this should not be a problem.

The other option is the use of an inducible promoter (other than AraC/pBad, promoter already used in our project in the targeting brick). The advantage of this option is that it gives the opportunity of lysing the bacteria at any moment of the process. If we model the filling of the Carboxysome and the rate in which reactions can occur inside it, then we would know exactly what is the moment to induce the autolysis.
Mechanism of the autolysis device.

Carboxysome disruption

Once the bacteria has released the Carboxysomes and we have purified them, the next step is to find a way for disassembling the shell proteins of the microcompartment and to obtain the content that it is needed. These purified samples can be stored up to 4 months at 4ºC with a protease inhibitor and still be possible to observe the geometrical shape of the Carboxysome (1). We have taken advantage of a method developed for the analysis of the enzymes inside the Carboxysome (RuBisCO and Carbonic Anhydrase) (1). In this method, the shell can be separated from its content using sonication for the gradual disruption of the shell proteins (2). We think that the same method could be adapted to whatever the content is inside the Carboxysome. The protocol is simple and quick,and includes the dilution of carboxysomes on buffers followed by sonication and centrifugation. The result obtained is the shell proteins as the pellet and the content as the supernatant.

A shorter alternative method for the disruption of the carboxysomes consist on freezing them for 30 minutes at -20ºC. This simple step will also allow us to recover the microcompartment content. Regardless the method chosen, the final result will be the release of the protein of interest. Even thought, this protein is now free from the Carboxysome, it is not a pure sample yet because of its fusion with the targeting sequences (BBa_K1113701) that delivers it to the microcompartment. Since this fusion could interfere with the activity of such proteins, we propose the use of a linker that could later be cleaved and separate the targeting sequence from the protein. There is a BioBrick (BBa_K316047) which is a linker that includes a protease cleavage site that could serve for this purpose. Another alternative is the use of Ph-sensitive linkers, like hydrazone linkers which are cleavaged at an acid pH (3) that will also separate the targeting sequence from the protein.
Diagram of the disruption of the Carboxysome
  • 1. Protein composition of the carboxysomes of Thiobacillus neapolitanus. Yolande A. Holthuijzen, Jan F. L. van Breemen, J. Gijs Kuenen, and Wil N. Konings.
  • 2. Activity of ribulose-l,5-bisphosphate carboxylase in intact and disrupted carboxysomes of Thiobacillus neapolitanus. Yolande A. Holthuijzen a, j. Gijs Kuenen b and Wil N. Konings
  • 3. Antibody-Drug Conjugates: Linking Cytotoxic Payloads to Monoclonal Antibodie. Laurent Ducry* and Bernhard Stump.