Part:BBa_K5242027
xylose-Inducible Component
1.Introduction
This part is a xylose-inducible element that produces elevated levels of Chk1 RNA when the external xylose concentration is elevated. It was designed for quantitative characterization of editing intensity.
2.Design
The VMA6 promoter at the forefront of this element is used to drive expression of xylRNLS.
Under low xylose concentrations, xylR can inhibit the transcription of the xylose promoter. However, when the xylose concentration increases, xylose binds to xylR, causing it to lose its repressive activity and thus promoting the expression of downstream genes.
Tsynth21 is a short synthetic terminator for use in Saccharomyces cerevisiae and Ribo00.S1 was designed to insulate upstream Tsynth21 from downstream pxyl.
The pxyl was initially a synthetic promoter with the recognition sequence of xylRNLS added in the middle, allowing the promoter to function only in the presence of xylose. [1]Subsequently, a study was made from here to construct a minimal version that responds to xylRNLS, and that optimized version is the one we use.[2]
The structure of the xylose-induced part is illustrated in Figure 1.
Figure 1.The structure of the xylose-induced part.
3.Experimental Characterization
3.1 Plasmid Construction and Yeast Transformation
We used the Gibson Assembly to build our plasmids. The sequencing results proved that the plasmid construction was successful.
3.2 Results of qPCR and FACs.
As Figure 2 shown, no clear relationship was observed between the target transcript concentration and editing efficiency. We hypothesized that this could be attributed to an insufficient induction period. Consequently, we planned to extend the induction time from 12 hours to 24 hours in subsequent experiments.
Figure 2. No clear relationship was observed between the target transcript concentration and editing efficiency.
However, in our next experiment, we were unable to detect red fluorescence under the microscope, suggesting a potential issue with homologous recombination, despite obtaining correct results in the colony PCR. Due to the extended duration of the experiment and the time constraints imposed by the start of the new semester, we were unable to repeat the experiment at this stage. Moving forward, we aim to obtain a more robust dataset in order to enhance the overall completeness of our project.
4.Referances
[1]Teo, W. S. & Chang, M. W. Bacterial XylRs and synthetic promoters function as genetically encoded xylose biosensors in Saccharomyces cerevisiae. Biotechnol J 10, 315-322 (2015).
[2]Chen, Y. et al. Genetic circuit design automation for yeast. Nature Microbiology 5, 1349-1360 (2020).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1786
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 502
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2273
Illegal BamHI site found at 3199 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1786
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1786
Illegal NgoMIV site found at 785 - 1000COMPATIBLE WITH RFC[1000]
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