Part:BBa_K5154000
GALp-lexAops
Galp-lexAops is a synthetic promoter that is modified from a standard Gal promoter in S. cerevisiae. In S. cerevisiae, Gal promoter is important for regulating the expression of genes responsible for the metabolism of galactose. It controls the expression of Gal gene family, which includes GAL1, GAL10 and other related genes.
In a standard Gal promoter, there is a UAS (Upstream Activation Sequence) located next to the promoter. When Gal4p is bound to UAS, the expression of downstream sequence will be switched on.
In Galp-lexAops, UAS is replaced by a lexAops-like structure which contains a lexA binding site. lexA is a bacterial repressor protein which is important in the SOS response. When lexA is bound to lexAops, the expression of downstream gene will be switched on. This synthetic promoter allows the construction of a lexA-dependent expression system.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 350
Illegal XbaI site found at 377
Illegal XbaI site found at 424
Illegal PstI site found at 462 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 350
Illegal PstI site found at 462 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 350
Illegal BamHI site found at 371
Illegal BamHI site found at 418
Illegal XhoI site found at 318
Illegal XhoI site found at 344 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 350
Illegal XbaI site found at 377
Illegal XbaI site found at 424
Illegal PstI site found at 462 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 350
Illegal XbaI site found at 377
Illegal XbaI site found at 424
Illegal PstI site found at 462 - 1000COMPATIBLE WITH RFC[1000]
Application in S. cerevisiae CWI pathway activation test
Introduction
The CWI (Cell Wall Integrity) pathway is crucial to the survival of S. cerevisiae in different environments, and it involves complicated enzyme cascades and signal transduction. In the cascade, activated mechanosensor wsc1 on the surface membrane interacts with a G-protein Rho1 and induces a MAPK cascade (Levin, 2011). Rlm1 is a transcription factor involved in the CWI pathway, and will be phosphorylated once the CWI pathway is activated (Levin, 2011).
Method
Plasmid Design
The plasmid was designed based on the lexA-Rlm1-lacZ reporter system (Watanabe et al, 1997). LexA is fused with â…” of Rlm1, and expressed constitutively under the control of an ADH1 promoter. LacZ is regulated by a synthetic reporter GALp-lexAops. When cells experience an osmotic pressure change, the CWI pathway will be activated. This leads to the phosphorylation of Rlm1, allowing the LexA-Rlm1 complex to bind the GALp-lexAops and activating LacZ expression. LacZ could therefore be used as a reporter for CWI pathway activation. The plasmid also contains a HIS3 marker, which carries a functional copy of HIS3
Figure 1 | map of pHPS100H constructed for S. cerevisiae CWI pathway activation test. Galp-lexAops is used as a promoter for lacZ reporter
Strains Used
Three different strains used: Y1, Y100H, HAS100L. Information about the strains is summarised into the table below:
Figure 2 | strains used in the CWI activation test
Y1 and HAS100L were obtained from Prof. Jürgen Heinisch, Universität Osnabrück. while Y100H was created by transforming Y1 with plasmid pHPS100H. Y1 has an elongated version of his-tagged wsc1 integrated into its genome, and HAS100L has a lexA-Rlm1-lacZ reporter integrated into its genome. Both strains have deficiency in synthesising histidine. Y100H was obtained by transforming Y1 with the plasmid pHPS100H (Straede et al, 2007), which contains a functional copy of HIS3 and allows cells to synthesise histidine.
β-Galactosidase activity test
The activation of CWI pathway was examined by testing the activity of β-galactosidase. β-galactosidase can hydrolyse X-gal ((5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) to form 5-bromo-4-chloro-3-hydroxyindole, which is further oxidised to form 5,5′-dibromo-4,4′-dichloro-indigo (Burn, 2012). The product formed is blue and insoluble. As a result, the activity of beta-galactosidase can be examined by adding X-gal and analysing the formation of the blue precipitate. X-gal stock solution was prepared by dissolving 40 mg of X-gal in 2 ml of DMSO to get a final concentration at 20 mg/ml. Cells were inoculated in YPD or Leu- His- Ura- drop-out medium to reach a OD600 value around 0.6. 6 ul of liquid culture was then added to the YPD plate as a single droplet, allowed to grow overnight. 5 ul of X-gal stock solution was added to the formed colony and incubated for an additional 30 mins. Blue colonies on the plate were then examined and recorded.
Results
Transformation of Y1 with pHPS100H
The transformant was picked from the positive plate and restreaked on the triple aa drop-out plate to ensure resulting colonies were true positive. The formation of colony indicates the presence of a functional copy of HIS3.The new strain was named Y100H.
Figure 3 | Transformation result
Validation with β-Galactosidase X-gal testing
The expression of lacZ was achieved by increasing the incubation temperature to 37 Celsius degrees to change the osmotic pressure around cells. Y1, HAS100L, Y100H was inoculated one night in YPD in advance to reach an OD600 at around 1.3, and was then diluted to 0.6~0.8. A YPD plate was divided into 3*3 areas. 6 ul of each strain was taken and added to the master plate. For each strain, three droplets were added in the same row. Allowing overnight incubation to form colonies on the plate. 5ul, 3ul, 1ul of X-gal stock solution was added to three colonies for each strain separately and incubated for an additional 30 mins.
Figure 4 | Activation test with X-gal
The colour change indicates lacZ was expressed by Galp-lexAops as a response to osmotic pressure change and CWI pathway activation. This suggests that Galp-lexAops is functioning and is under the regulation of lexA-Rlm1 in this particular construct.
Further Investigation and Magnetic Activation on Galp-lexAops controlled beta-galactosidase activity
We conducted further experiments to investigate the effect of incubation time / temperature will have on the activation of CWI pathway. Four masterplates were set up: 2 incubated at 30°C, and 2 incubated at 37°C. After one night, one plate was taken from both the 30°C and 37°C groups and treated with X-gal. The remaining plates were taken out and treated with X-gal after two nights. After two nights, we obtained results from the planned experiment.
Another test showed that for over-weekend incubation, 30 Celsius also showed more activation than 37 Celsius.
The result showed that for long-term incubation, 30 Celsius will have more CWI pathway activation than 37 Celsius. This may be explained by the fact that growth also activates the CWI pathway, and so, over a longer incubation period, better growth at 30°C outweighs the effect of temperature. It also emphasized that there is a noticeable level of background signal under standard conditions, highlighting the importance of controls in later experiments.
In magnetic activation experiments, HAS100L acted as a negative control because it contained the same reporter system for the CWI pathway but should not experience magnetic field-dependent CWI activation. 250 nm Ni-coated Magnetic nanoparticles (MNPs) and X-gal were added to colonies on two 35 mm plates, one colony each of Y100H and HAS100L. One plate was kept at room temperature, while the other was subjected to a low-frequency, high-strength magnetic field (100 mT, 1 Hz). Immediately after an hour of activation, a small amount of blue was visible around the edge of the activated colonies. Both plates were incubated at 30°C, and long-term incubation led to a more pronounced difference between the activated and non-activated plates.
The result showed that the activated group displayed more pronounced blueness compared to the non-activated group, indicates the magnetic activation is successful.
Conclusion
Through several rounds of testing, we successfully demonstrated that Galp-lexAops synthetic promoter works effectively as a part of the reporter system to CWI pathway activation, and it's strength was not significantly affected by heat or magnetic fields.
.References
1. Burn, Sally F. "Detection of β-galactosidase activity: X-gal staining." Kidney Development: Methods and Protocols (2012): 241-250.
2. Dupres, Vincent, Yves F. Dufrêne, and Jürgen J. Heinisch. "Measuring cell wall thickness in living yeast cells using single molecular rulers." ACS nano 4.9 (2010): 5498-5504.
3. Levin, David E. "Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signalling pathway." Genetics 189.4 (2011): 1145-1175.
4. Straede, Andrea, et al. "The effect of tea tree oil and antifungal agents on a reporter for yeast cell integrity signalling." Yeast 24.4 (2007): 321-334.
5. Watanabe, Yasuyuki, et al. "Characterization of a serum response factor-like protein in Saccharomyces cerevisiae, Rlm1, which has transcriptional activity regulated by the Mpk1 (Slt2) mitogen-activated protein kinase pathway." Molecular and cellular biology 17.5 (1997): 2615-2623.
//promoter
| biology | Saccharomyces cerevisiae |