Difference between revisions of "Part:BBa K4711050"

(→‎Usage and Biology)
(→‎Usage and Biology)
 
(One intermediate revision by the same user not shown)
Line 4: Line 4:
  
 
=Usage and Biology=
 
=Usage and Biology=
−
Due to the small amount of benzo (a) pyrene in kitchen waste oil, due to its strong carcinogenic ability, it has adverse effects on the environment and human health. Therefore, we designed a related circuit to degrade benzo (a) pyrene. At the same time, in order to achieve controllability of degradation, we designed a red light switch to control the expression of degradation enzymes. The gene circuit is shown below:
+
Transcription activation proteins can bind to specific sequences on DNA to initiate the transcription reaction of corresponding genes. The Binding Domain (BD) and Activation Domain (AD) are two independent domains on the transcription activation protein, and both of them are required for gene transcription activation. Based on this principle, yeast two-hybrid experiments have been designed to verify the interaction between the two proteins. Currently, yeast two-hybrid experiments have used two systems, LexA system and Gal4 system.
 +
 
 +
Following the design of the 2012TU_Munich team, we chose LexA as our DNA-binding domain. This is because, in contrast to GAL4-based systems, no knockdown of the yeast endogenous GAL4/GAL80 gene is required. It therefore also does not interfere with endogenous yeast metabolism and signaling systems because it recognizes only one specific prokaryotic DNA sequence, the so-called LexA binding site. Unlike the GAL4-based system, we do not need a special strain carrying a GAL4/80 deletion, so theoretically every yeast strain could work.
 +
 
 +
Meanwhile, based on the study of Oxana Sorokina et al. (2009), we found that the combination of PhyA and FHY1 had a faster and better response to red light. Therefore, PhyA and FHY1 were selected as the two main proteins in the red light system among the many phytochrome and its interaction factors.
 
<html>
 
<html>
  
 
   <figure>
 
   <figure>
  
−
       <img src="https://static.igem.wiki/teams/4711/wiki/design/d15.webp"width="100%" style="float:center">
+
       <img src="https://static.igem.wiki/teams/4711/wiki/design/d18.png"width="100%" style="float:center">
  
 
       <figcaption>
 
       <figcaption>
  
 
       <p style="font-size:1rem">
 
       <p style="font-size:1rem">
−
Fig 1 Benzo[a]pyrene degradation pathway
+
Fig 1 Different photosensitive pigments and their interacting factors
 
        
 
        
  
Line 21: Line 25:
 
   </figure>
 
   </figure>
 
</html>
 
</html>
−
Among them, phyA is a functional phytochrome receptor composed of an apolipoprotein and a phytochrome mobile protein (chromophore). Since chromophore cannot be synthesized in yeast, a similar compound, phycocyanobilin (PCB), purified from cyanobacteria, was added to the medium. PCB is readily taken up by yeast cells and is bound by the phytochrome apoprotein to form phytochrome photoreceptors.
 
  
−
Under red light (λmax = 660 nm) or far-red light (λmax = 730 nm), PhyA reversibly changes its conformation to be able to bind to FHY1, which binds PhyA to BD (DNA binding domain) and PHY1 to AD (transcription activation domain) via linker. The principle of yeast double hybrid was used to realize the expression of the target gene under the induction of red light.
 
−
<html>
 
  
−
  <figure>
 
  
−
      <img src="https://static.igem.wiki/teams/4711/wiki/design/d16.webp"width="100%" style="float:center">
 
  
−
      <img src="https://static.igem.wiki/teams/4711/wiki/design/d17.webp"width="100%" style="float:center">
 
−
 
−
      <figcaption>
 
−
 
−
      <p style="font-size:1rem">
 
−
Fig 2  Red light system schematic
 
−
     
 
−
 
−
      </figcaption>
 
−
 
−
  </figure>
 
−
</html>
 
 
===Source===
 
===Source===
  

Latest revision as of 12:18, 10 October 2023


Gal4(AD)+linker+Fhy1

Usage and Biology

Transcription activation proteins can bind to specific sequences on DNA to initiate the transcription reaction of corresponding genes. The Binding Domain (BD) and Activation Domain (AD) are two independent domains on the transcription activation protein, and both of them are required for gene transcription activation. Based on this principle, yeast two-hybrid experiments have been designed to verify the interaction between the two proteins. Currently, yeast two-hybrid experiments have used two systems, LexA system and Gal4 system.

Following the design of the 2012TU_Munich team, we chose LexA as our DNA-binding domain. This is because, in contrast to GAL4-based systems, no knockdown of the yeast endogenous GAL4/GAL80 gene is required. It therefore also does not interfere with endogenous yeast metabolism and signaling systems because it recognizes only one specific prokaryotic DNA sequence, the so-called LexA binding site. Unlike the GAL4-based system, we do not need a special strain carrying a GAL4/80 deletion, so theoretically every yeast strain could work.

Meanwhile, based on the study of Oxana Sorokina et al. (2009), we found that the combination of PhyA and FHY1 had a faster and better response to red light. Therefore, PhyA and FHY1 were selected as the two main proteins in the red light system among the many phytochrome and its interaction factors.

Fig 1 Different photosensitive pigments and their interacting factors



Source

Potential applications

References

[1]Sorokina, O., Kapus, A., Terecskei, K. et al. A switchable light-input, light-output system modelled and constructed in yeast. J Biol Eng 3, 15 (2009). https://doi.org/10.1186/1754-1611-3-15

[2]Hochrein L, Machens F, Messerschmidt K, Mueller-Roeber B. PhiReX: a programmable and red light-regulated protein expression switch for yeast. Nucleic Acids Res. 2017 Sep 6;45(15):9193-9205. doi: 10.1093/nar/gkx610. PMID: 28911120; PMCID: PMC5587811.

[3]Shimizu-Sato S , Huq E , Tepperman J M ,et al.A light-switchable gene promoter system[J].Nature Biotechnology, 2002, 20(10):1041-1044.DOI:10.1038/nbt734.

[4]Levskaya, A., Weiner, O., Lim, W. et al. Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461, 997–1001 (2009). https://doi.org/10.1038/nature08446

[5]Li, H., Qin, X., Song, P., et al. A LexA-based yeast two-hybrid system for studying light-switchable interactions of phytochromes with their interacting partners. Biotechnology Bulletin: English Edition, 2021, 2(2), 12. DOI: 10.1007/s42994-021-00034-5.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]