High levels of cellulose is the first module of our project. Thus, we have focused to cotransform two plasmids in E. coli BL21 (DE3) because it is resistant to toxic proteins. The firs plasmid is the operon bscABCD for cellulose biosynthesis while the second one is conformed by the genes Cmcax and CcpAx which are members of the cellulose synthase complex and allow an enhanced cellulose production rates.^4,5 Because the difficulties of chemical synthesis in four of the six genes, it was necessary to add aptamers at 5’ ands 3’ and the Gibson assembly, using psb1c3 as the host vector was delayed. To date, we have successfully assembled the plasmid containing the genes Cmcax and CcpAx.

Furthermore, we started the atomic force microscopy (AFM) analysis of the bacterial cellulose, analyzing its topography as the first step to approximate the total cellulose binding sites where the fusion protein will bind. This analysis will allow us to know the drug concentration in the cellulose.⁶

Two gBlocks were synthetized for in-frame protein assembly. The first gBlock consisting of lacI promoter, RBS and CDS of CBD cipA while the second gBlock containing the CDS of BMP2. Both of them were codon optimized for E. coli.

Because it has been demonstrated that the C-terminal domain of BMP2 provides osteogenic activity, ⁷ we intended to leave it free. Thus, it was necessary to fused it C-terminally to an N-terminal CBD.⁸ We decided to choose the CBD cipA because the higher affinity to bacterial cellulose compared to other CBDs that was reported by the team Imperial 2014.⁹

The CBD cipA has originally two linkers. The N-terminal linker is a signal sequence typical of the bacterial extracellular proteins while the C-terminal linker separating the CBD cipA from Coh3 module.

Then, in contrast to the team Imperial 2014, we decided to use only the C-terminal linker hypothesizing that this can provide a better binding strength than the use of two linkers.

Two gBlocks were synthetized for in-frame protein assembly. The first gBlock consisting of lacI promoter, RBS and CDS of CBD cipA while the second gBlock containing the CDS of BMP2. Both of them were codon optimized for E. coli.

Because it has been demonstrated that the C-terminal domain of BMP2 provides osteogenic activity,7 we intended to leave it free. Thus, it was necessary to fused it C-terminally to an N-terminal CBD. ⁸ We decided to choose the CBD cipA because the higher affinity to bacterial cellulose compared to other CBDs that was reported by the team Imperial 2014.⁹

The CBD cipA has originally two linkers. The N-terminal linker is a signal sequence typical of the bacterial extracellular proteins while the C-terminal linker separating the CBD cipA from Coh3 module. Then, in contrast to the team Imperial 2014, we decided to use only the C-terminal linker hypothesizing that this can provide a better binding strength than the use of two linkers.

ELP_C5 was obtained via overhang PCR using primers containing prefix and suffix in RFC25, and the plasmid pET-24a-ELP[V-150] as template.

Each ELP undergoes a Tt that leads to its aggregation or solubilization. The Tt of an ELP fusion protein is different to that of the free ELP depending of the surface accessible surface. Because we aimed a future clinical application of ELP fusion proteins in aggregated state, we decided to use ELP[V-150] with a Tt=28.2℃,¹⁰ lower than the corporal temperature. However we have cloning a 450 bp fragment of ELP[V-150] and we expect a similar Tt.