Part:BBa_K5226091
PRM-C34
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Introduction
One of the goals of iGEM SCUT-China-A is to use synthetic biology tools to build thermosensitive bio-switch for Halomonas TD. We successfully built one using ci857 regulatory protein and superfolder green fluorescence protein(sfGFP). For better performance and broader range of usage, we mutated CI857. Because Halomonas TD grew at 30°C in a mediocre manner, we sought out a narrower temperature region, the CI857 variant, TCI-42, and mutated it as well. We characterized the mutants together with PR-sfgfp and several version of well-performed T-switch were successfully obtained.
Usage and Biology
This is a composite part acting as a part of the T-switch we’ve created for Halomonas TD. CI857 is a widely used mutant of cI from bacteriophage λ as a thermo-genetic tool, exhibits strong repression on PR promoter at 30 °C by forming dimmer CI857 complex to achieve active and inactive transcriptional control of repressor and reporter under 37 °C and 30 °C, respectively. TCI is a temperature-sensitive variant of the bacteriophage λ repressor cI. It consists of N-terminal, C-terminal and the linkage fragment between them. It is a cold-inducible transcription factor. When the temperature is below 35℃, its activity will gradually rising. When the temperature is above 35℃, its activity will remain at a low level. The PRM promoter can be regulated by the native bacteriophage lambda proteins CI and CRO. PR promoter is a special promoter regulated by CI857. When CI857 formed dimer, it was inhibited, so that the following genes could not be normally expressed; The inhibition is released when the dimer is depolymerized, and then the gene is normally expressed. When PR-sfgfp was expressed together, we obtained a combination of thermosensitive bio-switch with better performances.
Experimental characterisation
Protocol
Strain construction
Because Halomonas TD is easy to lose plasmids, we converted plasmid into E. coli s17-1 and transferred plasmids to it by junction and then characterisd it.
Fluorescence Characterisation
-Add 1ml 60LB, 0.1% chloramphenicol and spectinomycin to the well, pick up the strain and add it to the hole, and then put the deepwell plate in the shaker at the corresponding temperature for 12-14 hours. Usually, for each strain we would prepare two wells in case of emergency.
-Add 1ml 60LB, 10μl bacterial fluid of last step,0.1% chloramphenicol and spectinomycin to the well, then put the deepwell plate in the shaker at the corresponding temperature for 12 hours.
-Take 100μl bacterial fluid of last step, add 400μl deionised water to the bacterial fluid, then centrifuge for 5 minutes, discard the supernatant and add 500μl 1X PBS, take 200μl into the enzyme standard plate to measure fluorescence using microplate reader（absorbance 600, excitation/emission wavelength 488/520）
Result
The result showed that the leakage of the selected mutants was all around 10. From the second one on the left, the fluorescence intensity are 100, 60, 55, 50. These selected variants further broaden the range of expression intensities of our T-switch, which means a wider range of applications.
References
[1]Feifei Li, Que Chen, Huaxiang Deng, Shumei Ye, Ruidong Chen, Jay D. Keasling, Xiaozhou Luo,One-pot selective biosynthesis of Tyrian purple in Escherichia coli,Metabolic Engineering,Volume 81,2024,Pages 100-109.
[2]Athina Vasileiadou, Ioannis Karapanagiotis, Anastasia Zotou,
Determination of Tyrian purple by high performance liquid chromatography with diode array detection,Journal of Chromatography A,Volume 1448,2016,Pages 67-72.
[3] Li, F., Chen, Q., Deng, H., Ye, S., Chen, R., Keasling, J. D., & Luo, X. (2024). One-pot selective biosynthesis of Tyrian purple in Escherichia coli. Metabolic Engineering, 81, 100–109.
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