Type I secretion system transports proteins in one step across the two cellular membranes [2]. It is composed of an inner membrane protein HlyB, a periplasmic channel protein HlyD and an outer membrane protein TolC. As every secretion system, secretion through this one too is mediated by the specific recognition of a signal sequence, which in this case are the sixty C-terminal amino acids of alpha-haemolysin HlyA. This sequence binds with the HlyB-HlyD complex and is then translocated into the channel [7].

There are several problems to address when secreting recombinant proteins through the Type I secretion system:

• First, the signal sequence HlyA is not cleaved when crossing the channel, but it needs to be cleaved to obtain a functional protein in the medium. That is why we fused the proNGF sequence with this sixty-amino acid long sequence, separated by the cleavage site for Tobacco Etch Virus protease (TEV). As we co-express TEV protease, the signal sequence will be eliminated once it is out the cell, and our proNGF can be active.
• Secondly, if TolC endogenously exists within E. coli, this is not the case of the transporter complex HlyB-HlyD. Thus, we co-transformed our bacteria with another plasmid pVDL 9.3, generously provided by Dr. Victor de Lorenzo, from Centro Nacional de Biotecnologia of Madrid, bearing HlyB and HlyD sequences, in order to get a chance to secrete NGF out of the cell. [8]

Since we imagined a technology which requires to integrate engineered bacteria in humans, we need to ensure that the engineered bacteria are contained in the specific environment they are designed for. That is why we thought of using a “kill switch” that cause them to die if they are released in the environment. Our kill switch is based on temperature: it enables bacteria to survive at human body temperature (37°C) but die at lower temperatures.

The kill-switch we use is based on a toxin/antitoxin combination, CcdB/CcdA [9]. CcdB is a lethal toxin for E. coli and its production is placed under the regulation of a temperature-sensitive promoter [9]. In permissive conditions, i.e. in human body, the expression of the toxin is repressed and the antitoxin is expressed at a constitutive low level in order to counteract any leaky expression of the toxin. This is done thanks to the modification of the Plac promoter, which is explained precisely in the KILL-SWITCH part of this wiki. When the temperature goes lower, the repression is lifted and toxin expression increases. The constitutive low level of antitoxin is no longer sufficient to counter the effects of the toxin, and the bacteria die.