Composite

Part:BBa_K3032051

Designed by: Denis Baronas   Group: iGEM19_Vilnius-Lithuania   (2019-10-15)


sfGFP tagged with light-inducible protein degradation tag (160AtLOV2, Ready for Expression)

Colight

This is a composite part including sfGFP (Bba_I746916) tagged with the light-inducible protein degradation tag (Bba_K3032001). sfGFP is under strong Anderson promoter (BBa_J23100) and strong RBS (BBa_B0034).


Introduction

Overview

There are plenty of two-component optogenetic systems that regulate transcription. However, such systems are not so responsive and robust as one-component. Not many one-component systems exist, so scientists do not have plenty of choices. Therefore, the regulation of bacterial functions is not usually as smooth as a scientist would like to.

Wouldn't it be nice to regulate and control bacteria functions directly in real-time and not affect the transcriptional level by messing up other endogenous bacteria functions? Well, a solution might be quite simple but not so easily achievable - a system that is based on protein degradation.

Such eukaryotic systems are available on the "market" [1], but what about our beloved prokaryotes? Recently, Cameron and colleges [2] evolved Mycoplasma protease (mf-Lon) based system, which might satisfy the demand of some scientists. However, neither similar eukaryotic nor solely mf-Lon based system provide one of the essential synthetic biology desire, that is, real-time control. So, we decided to design and create a system for prokaryotes where light works as a switch for protein degradation control.

Light-reactive LOV2 domain of Arabidopsis thaliana and Avena Sativa phot1 was chosen as an optogenetic protein, which acts as a switch [3] [4]. Meanwhile, mf-Lon, the protein responsible for degradation, was borrowed from Mycoplasma, which is orthogonal to E. coli [5] . It means that mf-Lon does not interact with endogenous E. coli components or degrade proteins tagged with endogenous E. coli degrons. Also, the degradation module of our system is small enough, so it should not alter native protein functionality.

Design

How does our system work? It easy, and it is simple.

Our light-driven degradation system consists of 2 cassettes. The first cassette contains IPTG inducible mf-Lon protease, which can recognize degron and degrade tagged protein. The second cassette consists of POI (protein of interest) fused to LOV2 domain, which has a tightly packed degron on the C terminus. By this mean, degron is sterically hidden and cannot be recognized by protease in the dark state. When bacteria are illuminated with 470 nm light, LOV2 domain’s C terminal alpha-helix unfolds, and the degron becomes no longer hidden from the protease. The unfolding is caused by the adduct formation of FMN and Cys residue of LOV2 [6] . After this moment, degron becomes accessible to the mf-Lon protease, which recognizes it and afterward degrades the whole protein.

An animation showing the mechanism of post-translational control.


Part Characterization

Experiments

First of all, we designed light-inducible protein degradation tags by fusing degron to the C-terminus of the AtLOV2 domain. In total, 11 light-inducible protein degradation tags were created, which differ in the AtLOV2 domain length. It is a critical point in the rational design of fusion proteins to detect the proper length of a functional domain to maintain its function. In our case, AtLOV2 should keep we ability to fold and unfold its C-terminus as the degron sequence is located in the C-terminus. Afterward, we fused these tags with POI, which, in our case, is sfGFP to test which degrons possessed the best results.

We began our experiment by letting the bacteria grow in the dark for 3h, both induced with IPTG and non-induced. Then, we measured their fluorescence levels and divided them with OD. The fluorescence/OD level was the same for all constructs (Figure 1).

Figure 1.The change of POI level after 3 hours in the dark. Growing temperature 30°C, Dh5ɑ strain.

Finally, we illuminated the same bacteria with 470 nm blue light, and after 24h, we measured the fluorescence. Several light-inducible protein degradation tags showed positive results. Tag, which consists of AtLOV2 length of 160 amino acid, showed a 40% decrease of steady-state level comparing to the non-induced of-Lon construct. (Figure 2).

Figure 2. The change of POI level after incubating the same bacteria in the 470 nm blue light, the measure was taken after 24 hours. Growing temperature 30°C, Dh5ɑ strain.


New possibilities

Our sophisticated mathematical model shows that light-regulated degradation opens the possibility to obtain any desired steady-state protein level. But for that purpose, a controller is a must. Therefore we developed one, so don’t miss the chance and take a look at our novel controller and mathematical modeling!

Final words

All in all, we developed a novel light-tunable prokaryotic degradation system and showed that rational protein design and functional coupling of proteins with different functions could lead to new optogenetic tools valuable for synthetic biology. Tunable protein degradation with the possibility to control protein steady-levels could become a very versatile and useful instrument for synthetic biology. Our light-tunable protein degradation system would give synthetic biologists a new tool for creating sophisticated gene networks without affecting or disrupting transcriptional levels in bacteria.

References

  1. ↑ Usherenko S, Stibbe H, Muscó M, Essen L-O, Kostina EA, Taxis C. Photo-sensitive degron variants for tuning protein stability by light. BMC Systems Biology. 2014;8(1):128. doi:10.1186/s12918-014-0128-9
  2. ↑ Cameron DE, Collins JJ. Tunable protein degradation in bacteria. Nature Biotechnology. 2014;32(12):1276-1281. doi:10.1038/nbt.3053
  3. ↑ Sakai T, Kagawa T, Kasahara M, et al. Arabidopsis nph1 and npl1: Blue light receptors that mediate both phototropism and chloroplast relocation. PNAS. 2001;98(12):6969-6974. doi:10.1073/pnas.101137598
  4. ↑ Kennis JTM, Crosson S, Gauden M, van Stokkum IHM, Moffat K, van Grondelle R. Primary Reactions of the LOV2 Domain of Phototropin, a Plant Blue-Light Photoreceptor †. Biochemistry. 2003;42(12):3385-3392. doi:10.1021/bi034022k
  5. ↑ Gur E, Sauer RT. Evolution of the ssrA degradation tag in Mycoplasma: Specificity switch to a different protease. PNAS. 2008;105(42):16113-16118. doi:10.1073/pnas.0808802105
  6. ↑ Kennis JTM, Crosson S, Gauden M, van Stokkum IHM, Moffat K, van Grondelle R. Primary Reactions of the LOV2 Domain of Phototropin, a Plant Blue-Light Photoreceptor †. Biochemistry. 2003;42(12):3385-3392. doi:10.1021/bi034022k






Sequence and Features


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1189
    Illegal SapI.rc site found at 74


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