Part:BBa_K5226089
PR/PRM-sfgfp
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 141
Introduction
One of the goals of iGEM SCUT-China-A is to use synthetic biology tools to obtain Halomonas strains that can produce tyrian purple. We chose to introduce four enzymes that is either necessary or beneficial to the production of tyrian purple. There were stth,fre,tnaA and fmo. Because both Stth and TnaA can utilize tryptophan, and tryptophan has a stronger preference for TnaA than for Stth, we introduced the thermalsensitive bio-switch that we built for Halomonas TD to separate the expression of the two enzymes to increase yield.
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). And for better performance and broader range of usage, we replaced the promoter of sfgfp, characterized together with PRM-ci857, and a thermalsensitive bio-switch with lower leakage was 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. 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 reporter genes sfgfp could not be normally expressed; The inhibition is released when the dimer is depolymerized, and then the following genes could normally expressed. By using both PRM and PR promoters simultaneously, the OR binding site is increased. When PRM-ci857(BBa_K5226063) was expressed together, we obtained a combination of thermosensitive bio-switch with lower leakage.
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 was reduced by 58% and the fluorescence intensity of sfgfp was controlled at around 40, which means this version of T-switch could be used to control genes with a desired gene expression intensity of around 40. For example, our composite part <a href="https://parts.igem.org/Part:BBa_K5226086">BBa_K5226086</a> and <a href="https://parts.igem.org/Part:BBa_K5226087">BBa_K5226087</a>
References
[1]Wang, X., Han, JN., Zhang, X. et al. Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli. Nat Commun 12, 1411 (2021). https://doi.org/10.1038/s41467-021-21654-x
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