Bacteria

Bacteria containing and actively expressing the injectisome system are pathogenic, meaning that they can make you sick. The system allows all kinds of different bacteria to shuttle disease-triggering proteins through the surface of our body and into our cells, which obviously makes you ill. The illness can vary somewhat from bacteria to bacteria, but the species that donated the injectisome-part we used - enteropathogenic Escherichia coli (EPEC) - triggers diarrhea in humans, most prevalent in children (1).

A better version

As such, the effects of that bacteria is unpleasant to say the least, and it's very much a must to to ensure the bacteria used in our Protein Printer is non-pathogenic. A system that produces pure disease-causing protein is near worthless, and the same can be said of a system that produces the target protein alongside disease-causing proteins. Our Protein Printer needs to produce only the wanted target protein, and nothing else for it to be functional.

In order to ensure this, we acquired a bacterial strain with the help of Luis Ángel Fernández Herrero (PhD, Principal Investigator, National Center for Biotechnology (CNB) Madrid, Spain), that expressed the EPEC injectisome in the non-pathogenic E. coli K-12 chassis.

This means that the useful injectisome has been separated from the disease-causing parts of the bacteria and is now functional in a non-pathogenic bacteria instead. This new and improved - for our purposes at least - EPEC bacteria was dubbed Synthetic injector E. coli (SIEC) (2)

The new SIEC bacteria has the advantages of having the injectisome system, but is not capable of making the natural disease-causing proteins alongside it. This strain is therefore way more suitable for our purpose of producing proteins made for actual medical purposes without the fear of other diseases!

The Injectisome

The injectisome (Type III secretion system) is a needle-like, protein structure used by some pathogenic bacteria (in our case E. coli) to inject effector proteins into other cells, for example in our gut, to make the environment more habitable for pathogenesis. For example, Injectisome-producing E. coli uses the injectisome to attach itself to the intestinal cells and inject these effector proteins into them to make them favorable for colonization (1).

The injectisome´s structure is in theory quite complex but simple to understand if one just thinks of a syringe, but a tiny molecular syringe. The base is made up of circle like structures which are both on the inside and outside of the cell membrane, to keep the injectisome in place. These circle like structures are connected together with a centrally located tube structure. On top of this base comes the needle structure which, on its, top has a, so called, tip structure, which aids in attachment and forming of the hole which toxins are injected through (2).

Injectifacts:
-The Injectisome translocates more than 25 effector proteins, which make the infected cell habitable
-The EPEC injectisome in our strain is one of the biggest injectisome reaching up to 700 nm 
-The SIEC strain we used can have a diameter of 0.68-1.0µm, which is about 70 times smaller than the thickness of your hair (3)

Chaperones

We expressed the chaperones CesF and CesT in our bacteria E. coli K-12, which are proteins that facilitate effector protein secretion in E. coli type-III secretion system. The chaperones are essential for making sure the injectisome is assembled correctly and for facilitating the unfolding of the target protein before it is secreted through the injectisome and across the membrane (2).

References:

(1) Ochoa, T. J., Barletta, F., Contreras, C., & Mercado, E. (2008). New insights into the epidemiology of enteropathogenic Escherichia coli infection. Transactions of the Royal Society of Tropical Medicine and Hygiene, 102(9), 852–856. https://doi.org/10.1016/j.trstmh.2008.03.017https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2575077/

(2) Ruano-Gallego, D., Álvarez, B., & Fernández, L. Á. (2015). Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells. ACS Synthetic Biology. https://doi.org/10.1021/acssynbio.5b00080

(3) Zaritsky A, Woldringh CL. Chromosome replication rate and cell shape in Escherichia coli: lack of coupling. J Bacteriol. 1978 Aug135(2):581-7PubMed ID355235