A LIVE BIOTHERAPEUTIC
Since GARGANTUA is planned to work as a live biotherapeutic, several facts should be considered to increase stability and safety of the chassis strain. Having in mind recent probiotic designed strains , FDA Guidance on Live Biotherapeutic Organisms (docket number FDA-2010-D-0500), and the concerns that emerged upon our discussions with specialists, we explored which is the best engineering approach to address this in the specific case of our probiotic. We discussed which steps could be added to our experimental design to pave the way for an appropriate medical implementation of our probiotic, coming up with the following.
Firstly, we aimed to circumvent the use of antibiotic resistance genes in our strain in order to avoid horizontal gene transfer of synthetic circuits from our therapeutical strain into the gut ecosystem. Secondly, we targeted chromosomal integration of the enhanced intake properties to provide our design with genetic stability and robustness of expression in the gut. Thirdly, we intended to make overexpression of beta oxidation genes inducible, eliminating the metabolic burden of protein synthesis during the growth phase. This is highly important in industrial large scale culture process, where this burden could diminish final yield.
We thought that Multiplex Automated Genome Engineering (MAGE)  technology could provide a ideal platform for achieving the aforementioned characteristics that a safe and efficient live biotherapeutic should incorporate. The reason that lead us to use MAGE was the fact that intake capabilities are already present in the metabolism of E. coli. Therefore, simply tuning their expression in order to achieve the desired result is needed. Moreover, MAGE is highly scalable, and can be implemented in probiotic strains such as E. Coli Nissle, which would allow for direct editing of strains with therapeutic potential.
How does MAGE work?
Multiplex Automated Genome Engineering (MAGE) is a directed evolution tool for multiple genetic loci based on the lambda phage homologous recombination abilities for targeted mutagenesis. Exo, beta and gam phage lambda genes are induced under the temperature-sensible $\lambda$ cI promoter which allows for temperature inducible expression of recombination machinery in MAGE strains . This expression promotes the recombination of electroporated ssDNA oligonucleotides targeting specific sites which can introduce deletions, mismatches or insertions. Multiple cycles of electro competence induction, electroporation, and population expansion make the editing process highly scalable. Remarcably, this process can be free of selection markers, which are not desired in biotherapeutical strains.
Genomic integration of LCFA uptake:
A simplistic albeit efficient approach consists in silencing the fadR gene, which is the main regulator of fad genes. Its absence should therefore raise constitutive levels of fad genes. To achieve this, we designed an oligo that when introduced in to the genome by the recombineering machinery would code for two stop codons in the beginning of the fadR coding sequence.
Considering the fact that beta oxidation genes are not solely regulated by the fadR repressor  we considered the introduction of T7 phage RNA polymerase promoter sequences upstream of fatty acid (FA) intake and degradation pathway genes. This would provide an orthogonal and inducible control of all the components in our system, which would be complicated to achieve merely by the use of synthetic constructs due to the large sequence size of the pathway. Genomic integration of the T7 polymerase is needed so as to achieve the desired levels of expression of the modified genes. The control of the polymerase under an inducible promoter provides the possibility of avoiding the metabolic burden caused by overexpression of beta oxidation genes in early stages of the probiotic strain production and batch culture. This would result in a more efficient production process.
Semirational Directed Evolution of uptake capabilities:
Incorporating sequence variation in to RBS of a particular pathway using MAGE can be used for optimization of expression of a particular metabolic pathway. In our case, this could be used for fine tuning the expression and consequently the efficiency of beta-oxidation. A minimal media complemented with LCHFA as the only carbon source could be used as a selection method for optimal RBS sequences and organisms that present enhanced uptake of LCHFA.
Oligo design & Cycling process:
The oligo of choice for silencing fadR was designed (using MODEST) so as to introduce two STOP codons within the coding frame of the gene. Glutamic acid 13 and 14 were mutated in order to induce STOP codons. Using the same in silico predictor, we designed recombinant oligos of 90bp in length to include T7 polymerase promoter sequences as described in the CoSelection mage .
LacZ silencing is used as a positive control when performing MAGE cycles due to its easily visible phenotype. Moreover, an improved alternative method for screening fadR mutants was designed. Our fatty acyl-CoA biosensor is dependant on fadR repressor levels, which acts on the pFadBA promoter sequence. We envisioned that transforming our part BBa_K2581006 into MAGE strain EcM2.1 could allow for a efficient screening of fadR knock-outs, since the absence of the regulator on the promoter should lead to the expression of basal levels of RFP (See More).
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 Wang, H. H., Isaacs, F. J., Carr, P. A., Sun, Z. Z., Xu, G., Forest, C. R., & Church, G. M. (2009). Programming cells by multiplex genome engineering and accelerated evolution. Nature, 460(7257), 894.
 Cho, B. K., Knight, E. M., & Palsson, B. Ø. (2006). Transcriptional regulation of the fad regulon genes of Escherichia coli by ArcA. Microbiology, 152(8), 2207-2219.
 Wang, H. H., Kim, H., Cong, L., Jeong, J., Bang, D., & Church, G. M. (2012). Genome-scale promoter engineering by coselection MAGE, 9(6). https://doi.org/10.1038/nmeth.1971