Nanotechnology for Synthetic Biology: Crossroads Throughout Spatial Confinement

International audienc

TEM imaging of RMM-10/11 pairs co-expressed with GFP1-9.

A, Wild-type RMM-10/11 pairs forms nanotubes when co-expressed recombinantly in E. coli with GFP1-9. Structures are however less well-defined than when studying the His-tagged RMM version. Honeycomb bundles reminiscent of those occurring with WT RMM are indicated with arrows. B & C, Assemblies might still occur in cells overexpressing combinations of sm-RMM-10/11 or dm-RMM-10/11 constructs, respectively, though structuration do not match to nanotubes or 2D-layers. D, Overexpression of tGFP partners based on the RMM triple mutant resulted in accumulation of proteins around cell poles, suggesting potential aggregation. Two images are presented for each mutant. E, Analysis of soluble protein fractions by SDS-PAGE. Gel-loaded material corresponds to soluble supernatants after centrifugation 21000 g of lyzed cells overexpressing WT RMM or mutant versions, in late phases of growth. Constructs carrying RMM in WT or mutants versions in fusion to His6 or GFP10/11 tags were analysed. All constructs studied in panels A-D, and on the right side of panel E, correspond to bicistronic single vectors with 30/27 linkers. (TIF)</p

Kinetics of nanotube formation in <i>E</i>. <i>coli</i>.

Observations by TEM of cells over-expressing His6-tagged RMM at different culturing moments after induction, indicated on the left. Longitudinal and transverse views are provided for each time point. An increase of nanotube density is evidenced at longer growth times. (TIF)</p

Influence of Linker length on tGFP reconstitution.

RMM were connected to GFP10 or GFP11 peptides with flexible linkers (Lk, rich in glycine/serine) of varied sizes. For each construct, the residue number of the GFP10-connecting Lk is given first, separated by a slash from residue number of the GFP11- connecting one. A, Maximal fluorescence signal according to Lk length. The 100% reference case is RMM with Lk30/27 (black bar). Lk30/27 were also present in BWI, CcmK3 controls. Empty corresponds to BL21(DE3) transformed with pET26b. B, Delay of fluorescence appearance. The calculated time differences between the midpoints of fluorescence and cellular growth are presented according to the residues number sum of the two Lk in the construct. BWI value exceeded 6h in delay of fluorescence appearance and is not shown here. C, Kinetics of tGFP partners expression depending on linker sizes. Cells expressing GFP1-9 and RMM pairs connected by indicated Lk to the GFP tags (pointed by arrows in panel B) were collected 2, 4 or 6h after induction. After cellular lysis, soluble fractions were analyzed by SDS-PAGE. The dashed line indicates 2 independent gels. The grey triangle on the right indicates GFP1-9 migration position. Molecular weights (kDa) of protein ladder components are on the left.</p

tGFP assay to monitor the oligomerization of BMC shell components.

A, tGFP reconstitution informs on homo-hexamer formation. Fluorescence values with pairs of indicated proteins organized in a bicistronic mode with Lk30/27 connectors. B, detection of association of BMC-P and BMC-T components. tGFP assay was performed similarly but on CcmL and CsoS4B BMC-P and on the CsoS1D BMC-T. Fmax presented in A and B panels are given as the percentage with regard to values measured for the reference RMM control (black bars). C, Verification of protein expression. Total protein contents extracted at late growth phases from panels A and B were analyzed by SDS-PAGE. White and black arrows point at presumable POI-10 and POI-11 bands, respectively. The asterisk notifies combinations of GFP10/11 N-ter-tagged monomers. See Fig 1 for other details.</p

Constant tGFP reconstitution in the presence of mutations impeding BMC-H assembly.

Fmaxvalues were estimated from fits to sigmoidal curves of data collected over 16 h, with induction from the culture start. In the case of BWI, CcmK3 and other cases with too low fluorescence values, fits were not reliable, and the value plotted corresponded to the highest fluorescence point over the course of the culture. Plotted values are given as the percentage with regard to the reference case: RMM-10/11 with Lk30/27 in an independent-transcript organization mode (black bar).</p

Diminished detection of protein bands when POI-10 or POI-11 are co-expressed with GFP1-9.

Protein expression levels were monitored by SDS-PAGE from BL21(DE3) transformed with single vectors, coding for separate tGFP partners as follows: pET26b coding for POI-10 on top, pACYC (11) on top for both GFP1-9 and POI-11, pET15b for POI-11, both in middle an, and pACYC (10) for GFP1-9 plus POI-10 in the middle. Shown are total contents of cells pelleted after 16 h cultures. (TIF)</p