E n g i n e e r i n g

Design

Our initial design involved a plasmid fusing the tyrosinase gene from Bacillus megaterium (Tyr1) with the AIDA (Adhesin Involved in Diffuse Adherence) system. This system was chosen for its ability to anchor protein domains to the outer membrane and expose the linked Tyr1 protein extracellularly. The host strain for this first expression attempt was the standard laboratory strain, E. coli K12 W3110.

Figure 2: Plasmid design for pAIDA-Tyr1.

Reference : Hörnström D, Larsson G, van Maris AJA, et al. (2019) Molecular optimization of autotransporter-based tyrosinase surface display. Biochimica et Biophysica Acta (BBA) - Biomembranes 1861: 486–494.

Build

The build phase was successful. The pAIDA-Tyr1 plasmid was assembled via megaprimer PCR and transformed into wild-type E. coli W3110. Positive clones were selected and sequenced to confirm the absence of mutations.

Tests

Clones were cultured in liquid and solid media induced with IPTG and supplemented with tyrosine and copper. A blackening of the medium, indicating melanin production, was expected.

Progress was monitored by visual observation, spectrophotometry at 405 nm, and Western Blot. The results were minimal: coloration was faint, absorbance was very low (figure 4), and Western Blots showed a protein of a smaller size than expected (Figure 5).

Enzymatic Assay Activity:

Figure 3 : tyrosinase activity measurements

In vivo tyrosinase activity measurements showing the strong increase in absorption at 400 nm after incubation of bacteria with IPTG, tyrosine and Cu2+ in W3110 bacteria containing our pAIDA1-Tyr plasmid after about 10 hours, and in the Delta OmpT strain (which grew less well) after about 30 hours.

Learn

The results suggested instability of the fusion protein. Literature indicates that the protease OmpT, present in the outer membrane of E. coli K12, is often responsible for such cleavage. OmpT is an endoprotease known to specifically cleave linker domain sequences in surface-displayed proteins, making the AIDA-Tyr1 construct a prime target. This led us to our second cycle. Instead of a laborious and potentially detrimental redesign of our construct to remove cleavage sites, we opted for a more efficient host engineering strategy.

Design

To overcome the barrier of proteolytic degradation, we designed a second cycle using the same W3110 strain, but genetically optimized by deleting the ompT gene. This directly removes the primary source of Tyr1-AIDA degradation.

Build

The build phase for this cycle was identical to the first: the pAIDA-Tyr1 plasmid was transformed into the new E. coli W3110 ΔompT host.

Tests

Tests were repeated under the same conditions as before. This time, the results were clear: the medium turned intensely black, indicating strong melanin production. Absorbance measured by spectrophotometry was significantly higher. Most importantly, the Western Blot showed a single, intact protein band at the expected size, with no evidence of cleavage.

Figure 4: Results with the optimized strain ΔompT vs with the WT

Figure 5: Western Blot results confirming intact protein.

(Lane M: Protein marker. Lane T-: Negative control (pAIDA W3110 ΔompT). Lanes 1', 3', 4': Colonies of pAIDA-Tyr1 W3110 ΔompT showing the full-length protein at ~97 kDa. The expected size of the AIDA-Tyr1 fusion protein is 97 kDa; the control and marker confirm the specificity of the result.)

Figure 6 : schematic representation of melanin production results between the WT and deltaompT strains

Learn

We learned that the performance of a genetic system is inextricably linked to the physiological context of its host. For the export, secretion, or surface display of proteins, it is critical to select mutant strains lacking proteases that can interfere with production. Our host engineering approach successfully resolved the primary bottleneck, validating a core principle of synthetic biology.