Part:BBa_K5242033
safety_lock_Hsp26_GSDMD
1. Introduction
This part is the core part of our Strain Security System, modified from sensor_Chk1. After importing this part, when the yeast is cultured under unusual conditions, the expression of the suicide gene will cause the yeast to die; while when the yeast is cultured under high-temperature conditions, the module will be automatically removed without negatively affecting the growth of the yeast.
2.Design
The model of the Strain Security System is depicted in Figure 1. Additionally, this component is illustrated in Figure 2. Regarding the ogRNA, ogRNA_HSP26 was selected, and the suicide gene chosen for this system was GSDMD'N.
Figure 1.The model of Strain Security System
Figure 2.The structure of this part.
We chose a galactose-inducible promoter for this study because we assumed that the key genes, which the security system is intended to protect, would also need to be induced by galactose to be activated. When the protected gene is induced and expressed, the security gate element is simultaneously expressed, and it triggers cell death in the yeast if the culture conditions are not appropriate. If the protected gene is induced using a different promoter, then the security system should also be equipped with a corresponding inducible promoter.
2.1 Backbone
In our design, we incorporated the Cre recombinase gene downstream of the sensor and designed loxP sequences on either side of the suicide gene. Under specific conditions like heat stress, the concentration of stress response gene transcripts is high, which can form duplex with sensor. thus, ADAR is able to edits the stop codon of the ogRNA sequence in the sensor, allowing for the expression of Cre recombinase. The Cre recombinase can knockout the suicide gene, preventing the death of the strain. When conditions change, Cre recombinase is not expressed in large quantities, so the suicide gene is continuously expressed and accumulates, eventually leading to the death of the strain. This method avoids the drawbacks of persistent leakage expression of the suicide gene, which could otherwise damage the cells. The mechanism above is shown in Figure 3.
Figure 3.The mechanism of this part.
2.2 Endogenous Stress Response Gene
HSP26 transcripts are undetectable in unstressed cells but are strongly induced by heat shock, salt shock, cell cycle arrest, nitrogen starvation, carbon starvation, oxidative stress, and low pH. [1][2][3]Under these stress conditions, HSP26 expression is upregulated by the transcription factors Hsf1p and Msn2p/Msn4p, which bind to the heat shock elements and stress response elements in the HSP26 promoter, respectively, which made it a suitable target for our security system. When fermented at 40°C for 6 hours, HSP26 expression levels are upregulated by 166-fold compared to those at 30°C. This indicates that HSP26 is significantly upregulated under high-temperature conditions to protect cells from heat damage.[4]
ogRNA_HSP26 is the part we designed for monitoring HSP26. It can be complementarily paired with Hsp26 and contains an A-C mismatch site in the middle for the introduction of ADAR editing. The addition of four of these MS2 sequences facilitates ADAR recognition and editing. At the beginning and end of the fragment, we added E2A and LV2A peptides, respectively, followed by GSG linker, allows the proteins on either side to be separated.
Figure 4.Relative Transcription Levels of Saccharomyces cerevisiae Genes During Fermentation at 40°C
2.3 Suicide Gene
Gasdermin-N is a protein that induces pyroptosis in cells, with its domain divided into N-terminal and C-terminal regions. The N-terminal region possesses inherent pyroptosis-inducing activity and is generally believed to aggregate on the cell membrane to form pores, leading to cell pyroptosis. The C-terminal region can inhibit the activity of the N-terminal region; only when the N-terminal and C-terminal regions are separated can pyroptosis be triggered[5].
We decide to let yeast cell express the N-terminal domain of GSDMD to achieve its suicide. Experiments have demonstrated that introducing the N-terminal region of GSDMD into yeast cells severely affects their growth[6].
3. Experimental Characterization
3.1 Plasmaid construction
As before, we attempted to assemble this batch of plasmids using a multi-fragment Gibson Assembly. The Electrophoretic results of our plasmaids are as follows.The results showed that a portion of the plasmids were not constructed successfully。
Figure 5.The Electrophoretic Results of pSensor-URA-Cre plasmid and others
However, since we needed to construct the plasmid through six-fragment Gibson assembly, it was difficult and had a low success rate. At the same time, the laboratory needed to be renovated, which resulted in insufficient experimental time, so we were ultimately unable to complete the construction of all plasmids. We learned a lesson from this and tried to avoid six-fragment Gibson assembly in subsequent experiments.
4 References
[1]Burnie, J. P., Carter, T. L., Hodgetts, S. J. & Matthews, R. C. Fungal heat-shock proteins in human disease. FEMS microbiology reviews 30, 53-88 (2006). [2]Carmelo, V. & Sá-Correia, I. HySP26 gene transcription is strongly induced during Saccharomyces cerevisiae growth at low pH.FEMS microbiology letters 149, 85-88 (1997).
[3]Amorós, M. & Estruch, F. Hsf1p and Msn2/4p cooperate in the expression of Saccharomyces cerevisiae genes HSP26 and HSP104 in a gene‐and stress type‐dependent manner.Molecular microbiology 39, 1523-1532 (2001).
[4]Chen, Q., Fang, Y., Zhao, H., Zhang, G. & Jin, Y. Transcriptional analysis of Saccharomyces cerevisiae during high-temperature fermentation.Annals of microbiology 63, 1433-1440 (2013).
[5] Ding, J.et al. Pore-forming activity and structural autoinhibition of the gasdermin family.Nature 535, 111-116 (2016).
[6] Valenti, M., Molina, M. & Cid, V. J. Human gasdermin D and MLKL disrupt mitochondria, endocytic traffic and TORC1 signalling in budding yeast.Open biology 13, 220366 (2023). Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 781
Illegal PstI site found at 1138 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2406
Illegal PstI site found at 781
Illegal PstI site found at 1138 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2795
Illegal XhoI site found at 2988 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 781
Illegal PstI site found at 1138 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 781
Illegal PstI site found at 1138
Illegal AgeI site found at 409
Illegal AgeI site found at 2481 - 1000COMPATIBLE WITH RFC[1000]
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