Part:BBa_K5087025
Chromoprotein Reporter Unit: tsPurple
Introduction
This composite expression unit enables the production of the tsPurple chromoprotein in bacteria, which gives them a vivid purple 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 tsPurple 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, tsPurple absorbs visible light, giving a deep purple color to the cells in which it is expressed.
Chromoproteins offer several advantages over fluorescent proteins. The intense color of tsPurple allows for easy visual detection without the need for UV light, minimizing background fluorescence and avoiding UV-induced damage.
Our tsPurple 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 tsPurple unit a perfect fit for this application.
Design
In designing the reporter unit for the SynLOCK system, we considered several key factors:
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Visibility: The reporter needed to be easily visible to the naked eye, which led us to choose a chromoprotein over a fluorescent one.
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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.
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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].
Figure 1. Schematic representation of the parts used to design the reporter unit.
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 purple color visible to the naked eye.
Figure 2. Results of the transformation with the Golden Gate reaction mixtures after 17h incubation at 37°C.
Conclusions: The chosen plasmid is a medium copy plasmid, which is why the color is developing slower. The available data indicated that a 24-48 hour incubation may be needed for the color to be fully developed1.
It is worth noting that we tested four different reporter units in this experiment (with CDS of tsPurple (TU5), ts Purple with promoter BBa_J23116 (TU4), TannenRFP (TU3), and aeBlue (TU7) and the one presented on this page performed the best. The color was visible just 17h after plating, which distinguished this reporter from other ones we tested.
Figure 3. 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 TU5 corresponding to the composite part presented on this page.
Conclusions: A higher copy plasmid would improve color formation.
- TU5 was the unit that developed color the fastest during incubation and produced the highest intensity among all the units we tested. TU7 (BBa_K5087026) 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 4. 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 5. Pellets formed after centrifuging the overnight cultures carrying various transcription units (indicated in the picture).
Conclusions: The most promising reporter for our system proved to be the one described on this part page. It showed color in the shortest time after plating, which is advantageous for speeding up lab work. The color intensity was also satisfactory, remaining visible to the naked eye even when the reporter was inserted into a medium-copy plasmid. We decided to proceed with this reporter for our further lab work, choosing it as the primary reporter for our SynLOCK system.
Performance within the SynLOCK system
We proceeded to use this Composite Part within our system for crRNA synthesis. As such, this part became a building block of our system’s most key element — the SynLOCK Cassette (Learn more here).
Figure 6. Schematic representation of the SynLOCK system: The reporter in the Cassette is replaced with a crRNA spacer, allowing for straightforward visual screening of colonies that have accepted the spacer.
Our Cassette was tested in two plasmid backbones: PSB1C5C (indicated as SVR—System Vector Reporter) and pSB1C3 (indicated as USV—Ultimate System Vector). You can read more about the choice of those backbones on Cassette's part page (BBa_K5087017).
Figure 7. Results of obtaining SVR plasmid-carrying colonies after an overnight incubation at 37°C.
Conclusions: All colonies were visibly violet. The used plasmid backbone (PSB1C5C) is a high copy plasmid, therefore intense color was visible in the morning after just one night of incubation.
For better visualization, we spread the bacteria on a plate.
Figure 8. SVR carrying clones.
Figure 9. Overnight cultures of SVR colonies.
Conclusions: The chromoprotein production in SVR vectors is very high — the color is easily observed after an overnight incubation on a plate and in a liquid culture as well, which was not the case in previous cultures. This is the first time we observed color in a liquid culture without having to centrifuge it. The high-copy plasmid backbone we chose for this assembly helped us maximize color formation, which was the key goal for our system.
Figure 10. a) The transformation results of the ligation reaction creating the USV plasmid visualized against a white background and b) purple background. c) the negative control.
Conclusions: The ligation reaction was successful, as indicated by the purple color of the colonies, which confirms that the reporter was ligated correctly.
Sequence
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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
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