Part:BBa_K5087026

Chromoprotein Reporter Unit: aeBlue

Introduction

This composite expression unit enables the production of the aeBlue chromoprotein in bacteria, which gives them a vivid blue color. This unit was created as a reporter for SynLOCK — the crRNA Synthesis System for SHERLOCK, designed, built, and tested by the iGEM JU-Krakow team to simplify and standardize the process of generating custom crRNA. Learn more here.

Biology & Usage [1]

The aeBlue protein is a type of eukaryotic chromoprotein (CP), similar to the green fluorescent protein but distinct in its mechanism. Unlike fluorescent proteins that emit light when excited, aeBlue absorbs visible light, giving a deep blue color to the cells in which it is expressed.

Chromoproteins offer several advantages over fluorescent proteins. The intense color of aeBlue allows for easy visual detection without the need for UV light, minimizing background fluorescence and avoiding UV-induced damage.

Our aeBlue expression unit is ideal for applications that require visual markers without the need for the complexity of fluorescent detection systems. For example, in our SynLOCK system, we prioritized user-friendliness, making the aeBlue unit a perfect fit for this application.

Design

In designing the reporter unit for the SynLOCK system, we considered several key factors:

• Visibility: The reporter needed to be easily visible to the naked eye, which led us to choose a chromoprotein over a fluorescent one.

• Constitutive Expression: The reporter needed to be expressed constitutively, ensuring that there is no ambiguity about its presence in the cell. That is why we chose a constitutive promoter.

• Color Intensity: To achieve a more intense color, we opted to place the reporter in a high-copy plasmid.

Our choice of the BBa_J23110 promoter was significantly influenced by the results obtained by Liljeruhm et al [1].

Experimental Validation

Assembling the Composite Part

Chassis

This composite part was tested in both TOP10 (Thermo Fisher) and NEB® 5-alpha (New England Biolabs) E.coli strains. We chose these specific strains because they are widely accessible to iGEM teams. The 5-alpha cells were provided to us by iGEM’s sponsor, New England Biolabs, and the TOP10 cells were commonly used in our lab. We optimized the protocols for transforming these cells with our constructs, which can be found here for the TOP10 cells and here for the NEB 5-alpha cells.

DNA source and Backbone specifications

To construct this composite part, we utilized components provided in the 2024 iGEM Distribution Kit. For assembly, we followed the iGEM Type IIS (RCF1000) standard. Specifically, we employed the pJUMP29-1A(sfGFP) vector from the Distribution Kit as our plasmid backbone. This vector was chosen because it conforms to the LVL1 plasmid backbone specifications required by the Type IIS assembly standard. Initially, the backbone contained a green fluorescent protein (GFP) (BBa_J428326) reporter, as supplied in the Kit .

Note: For this experiment, we chose a medium-copy plasmid backbone to avoid overburdening the bacteria. We understood that this choice might result in slower color development. However, this was not a concern for us because the final destination of this reporter would be within our SynLOCK Cassette. This cassette is intended to be placed in a high-copy plasmid to ensure maximal crRNA yield.

The Golden Gate Assembly

We conducted a Golden Gate assembly reaction using our protocol (available here), integrating the four basic parts with the pJUMP29-1A plasmid backbone. This process resulted in excising the GFP module and substituting it with our composite part. Consequently, the bacteria lost the green fluorescence and instead gained a blue color visible to the naked eye.

It is worth noting that we tested four different reporter units in this experiment (with CDS of tsPurple (TU5), tsPurple with promoter BBa_J23116 (TU4), TannenRFP (TU3), and aeBlue (TU7). Only TU5 showed color after just 17h after plating.

Figure 1. Results of the transformation with the Golden Gate Reaction II mixtures after 17h incubation at 37°C. The respective transcription units are indicated in the image, with TU7 corresponding to the composite part presented on this page.

Conclusions: A higher copy plasmid would improve color formation.

• TU5 (BBa K5087025) was the unit that developed color the fastest during incubation and produced the highest intensity among all the units we tested. TU7 also showed potential.
• As anticipated, the negative control showed a few colonies exhibiting green fluorescence — those were the reclosed destination plasmids.

The observations were repeated 30 hours after plating.

Figure 2. Plates with colonies carrying different transcription units (indicated in the picture) 30h after plating.

All four overnight cultures prepared from colonies picked from the plates were centrifuged.

Figure 3. Pellets formed after centrifuging the overnight cultures carrying various transcription units (indicated in the picture).

Conclusions: We chose BBa K5087025 as the primary reporter for our system, however, we leave the one presented on this page as an alternative for future iGEM teams who would be interested in switching reporters within our system to tailor it to their needs.

Sequence

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Biosafety

We used the Asimov's tool — Kernel — to check the sequence's safety with the Biosecurity Sequence Scanner. The results showed no flagged sequences, confirming that this part is safe to use.

References

[1] Liljeruhm J, Funk SK, Tietscher S, et al. Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. J Biol Eng. 2018;12:8. doi:10.1186/s13036-018-0100-0