Part:BBa_K5242035

safety_lock_Hsp26_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 BAX-20.

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

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]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]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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1276
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2199
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2588
  Illegal XhoI site found at 2781
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1276
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1276
  Illegal AgeI site found at 409
  Illegal AgeI site found at 2274
  
• 1000
  COMPATIBLE WITH RFC[1000]