Part:BBa_K5242038
safety_lock_GLC3_Baxmyc
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.
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.
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.
2.2 Endogenous Stress Response Gene
GLC3 is the gene encoding 1,4-α-glucan-branching enzyme in Saccharomyces cerevisiae. The gene is located at V:133,120 - 135,234 and is involved in the glycogen biosynthesis process. [4]It adds branches to glycogen molecules. [5]GLC3 mRNA begins to accumulate when environmental glucose decreases to approximately 50% and peaks when environmental glucose is depleted, similar to other glycogen metabolism genes.[6]
2.3 Suicide Gene
The apoptosis regulator BAX plays a role in the mitochondrial apoptosis pathway. Under normal conditions, BAX is largely cytosolic due to continuous retro translocation from the mitochondria to the cytosol mediated by BCL2L1/Bcl-xL, thereby preventing the accumulation of toxic BAX levels at the mitochondrial outer membrane (MOM).[5] Under stress conditions, BAX undergoes conformational changes that lead to its translocation to the mitochondrial membrane, resulting in the release of cytochrome c, fragmentation of the interconnected mitochondrial network, and subsequent triggering of apoptosis.
Although full-length Bax protein can hardly induce apoptosis in yeast, it can be functional with minor modifications. Previous reports indicate that C-terminal c-myc-tagged human Bax is highly effective at killing yeast, and this cell death is accompanied by the release of cytochrome c.[6][7][8] We called it BAX_myc here.
3. Experimental Characterization
3.1 Plasmaid construction
As before, we attempted to assemble this batch of plasmids using a multi-fragment Gibson Assembly. The sequencing results of pSensor-URA-Cre plasmid are as follows.
Figure 5.The sequencing results of pSensor-URA-Cre plasmid are as follows
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]UniProt: the Universal Protein Knowledgebase in 2023
[5]Priault, M., Camougrand, N., Kinnally, K. W., Vallette, F. M. & Manon, S. Yeast as a tool to study Bax/mitochondrial interactions in cell death.FEMS Yeast Research 4, 15-27 (2003).
[6]Priault, M.et al. Investigation of the role of the C-terminus of Bax and of tc-Bid on Bax interaction with yeast mitochondria.Cell Death & Differentiation 10, 1068-1077 (2003).
[7]Greenhalf, W., Stephan, C. & Chaudhuri, B. Role of mitochondria and C‐terminal membrane anchor of Bcl‐2 in Bax induced growth arrest and mortality in Saccharomyces cerevisiae.Febs Letters380, 169-175 (1996).
[8]Clow, A., Greenhalf, W. & Chaudhuri, B. Under respiratory growth conditions, Bcl‐x (L) and Bcl‐2 are unable to overcome yeast cell death triggered by a mutant Bax protein lacking the membrane anchor.European journal of biochemistry 258, 19-28 (1998).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1276
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2199
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2588
Illegal XhoI site found at 2781 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1276
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1276
Illegal AgeI site found at 409
Illegal AgeI site found at 2274 - 1000COMPATIBLE WITH RFC[1000]
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