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This section provides a short summary of our main experimental achievements.

Nanoparticles:

We successfully synthesized different batches of nanoparticles, whose mean diameter is in a range between 200 nm and 300 nm. We ended up with seven different collections of samples: simple naked gelatin nanoparticles, simple biotinylated nanoparticles, CY5-labeled biotinylated nanoparticles, rGFP loaded nanoparticles (naked and biotinylated) and FITC-Dextran loaded nanoparticles (naked and biotinylated). All of them were characterized by DLS. These experiments show that two cargo transport strategies are possible: an external labeling (the one we used with CY5) or an internal loading (with FITC-Dextran and rGFP).

Figure 1: DLS experiment results of the first nanoparticles we obtained: their mean diameter is a bit below 200 nm.

Figure 2: ELISA-like assay. The high absorbance in wells B and H (containing biotinylated nanoparticles) was quantitatively detected using a plate reader. It showed that the nanoparticles were well biotinylated.

Figure 3: Confocal microscopy image showing outer CY5 labeling of the nanoparticles. The size of those nanoparticles is around 200 nm, which corresponds to the previous DLS characterization.

Figure 4: Fluorescent microscopy of the FITC-Dextran loaded nanoparticles. In contrast to the rGFP-loaded ones, their cargo stay inside. Those nanoparticles have been successfully characterized and biotinylated.

Figure 5: Fluorescent microscopy of GFP-expressing E.coli and nanoparticles. The bacteria were chemically labeled with streptavidin as a surrogate of the cell-surface display strategy. We then mixed them with biotinylated CY5-labeled nanoparticles. This merge image shows that the coupling is successful and that the nanoparticles are attached to the E.coli.

Cell surface expression:

Characterization of an existing part: The part BBa_K523013 (INP-YFP construct to export YFP at the membrane) had been characterized only by a comparison of pellet and supernatant fluorescence after centrifugation. We wanted to characterize it better to be sure that it was expressed on the outer membrane. We successfully showed that the YFP-INP was expressed at the membrane.

This fluorescence microscopy image shows that YFP direct excitation signal colocalizes with YFP-antibodysignal, meaning that the protein is not only at the membrane, but at the outer one.

Figure 5: INP-YFP expressing cells A)detection by WFP excitation (514nm); B) detection by biotinylated anti-YFP antibody and avidin daylight (650nm); C) Merged images show colocalization, proving that the fusion protein is expressed at the outer membrane.

Expression of streptavidin at the cell surface: Gibson assemblies of the three different streptavidin worked perfectly and sequencing results matched with what expected. The growth curve of transformed E.Coli showed delayed growth, but bacteria still divide with an acceptable rate. The assay with a fluorescent biotin supposed to bind streptavidin gave some positive results (some bacteria appeared flurescent when excited at the corresponding wavelenght) but since the negative control also showed fluorescence, nothing could be proved. However, a western blot anti streptavidin showed bands at the expected size of streptavidin, proving with certainty its expression.

Streptavidin export at the outer membrane was also needed to achieve our project in order to couple nanoparticles to bacteria. Three streptavidin were used to increase the success chances. The three were amplified and inserted into the Biobrick Bba_K523013 insted of YFP. So streptavidin was fused with INP supposed to export it at the outer membrane. The sequencing results of the Gibson Assembly were fine and proved that streptavidin was well in frame with INP in the three cases. The functionnal assays were less promising. An assay was performed with antibody anti streptavidin but showed no significant results. Biotin was then used because of its smaller size and showed .... on va le refaire demain/aujourd'hui. A Western Blot was performed with a lot of unspecific noise, still showing some bands at the expected size of INP-streptavidin fusion (53KDa).

Sensing:

For sensing part, two pH-dependent and a constitutive promoter were inserted in front of a superfolded GFP sequence (Biobrick BBa_I746916 ). The inserts, consisting in the three different promoters, as well as the insert were isolated and amplified successfully by PCR. A gel was made to assess it and the sizes were the expected ones. The Gibson assemblies went fine and the results matched 100% with the expected sequences. Fluorescence measurements were not really relevant since a lot of cells died in acidic pH, supposed to activate the promoter. However, even if we were not able to prove that acidic pH triggers expression of GFP, fluorescence could be seen, thus the promoters are active. Constitutive promoter showed nice results, which allowed us to deliver it in the kit (see outreach -> kit).

Effector:

With the exception of MMP9, Gibson assemblies worked fine. The sequencing results showed a STOP after MMP2 and gelE, but there was still a chance for them to be expressed, even whithout GFP. The Western Blot anti-His tag was negative even though His tag was supposed to be before STOP. However, it may be hidden in the protein. An assay to purify the two enzymes MMP2 and gelE showed the presence of the proteins by comparing the assay of arabinose induce (expressing enzymes due to the arabinose induced promoter) and non arabinose induced protein.

For triggering expression of degradating enzymes, arabinose sensitive promoter was chosed. The enzymes to insert were MMP9, MMP2 and gelE, the three degrading gelatin. The part BBa_I746908 was the backbone consisting in GFP driven by pBad promoter. The enzymes to insert would be either put instead of GFP or in addition to GFP. All final plasmids would have a His-tag to purify it easily. Sequences of the enzymes and the backbones were correctly PCR amplified. The Gibson were successfull for the MMP2 and gelE, but cells transformed with MMP9 didn't give any colony. Sequencing results of the other plasmids showed a stop codon between enzymes and GFP, the reason why they didn't appear green. However, the enzymes preceding GFP could still be expressed and an His-Tag purification was achieved since His-tag is placed before the stop codon. The different fractions were collected and analyzed on a SDS page :

Overall:

We can say that except minor problems, cloning succedeed well. The Gibson assemblies globally worked out and we had no trouble growing the resulting transformed bacteria. The most delicate part was the characterization of our parts with functionnal assays. However, a lot of parts showed encouraging results but would maybe need to be studied in more detail. The naoparticles is the part that worked out well, nanoparticles could be synthetized and loaded. This project was ambitious and was almost achieved, and we are really proud of sharing our results with the iGEM community!