Part:BBa_K2625001

Secretion factor alpha pro + MYR1 myrosinase

Myrosinase belongs to a family of enzymes involved in the plant defense. It can catalyze the hydrolysis of glucosinolates. The final product that derives from this hydrolysis has been proved to inhibit carcinogenesis and tumorigenesis in certain circumstances.

Figure 1: Plasmid design with Alphapro and Myrosinase(Myr1), the spacer and the Kozak sequence. The plasmid of origin is from pSB1C3.

Characterization

This part was developed by our team to be used as a way to kill colon cancer cells. The Myrosinase itself is coupled to a secretion peptide since it needs to be present in the media for it to interact with the glucosinolates. A Kozak sequence was also introduced at the begining of the protein to enhance expression.

To assess if the protein was secreted and functional the yeast was allowed to grow overnight. After cultivation the expression of myrosinase was induced via addition of alpha-pheromone, since the promoter that was used, FUS1, only enables transcription when alpha-pheromones are present. The yeast was incubated with the pheromones for two hours, after that the culture was filtered to remove all cells but retain the myrosinase in the resulting cell free solution. The solution was split into two tubes and sinigrin, a glucosinolate, was added to one of the tubes.

Prior to this a 24-well plate with RKO cells was grown to about 80% confluence in EMEM 10% FBS media. The myrosinase solution with sinigrin was added to six wells and the solution without the substrate was added to six different wells. The cancer cells were incubated for an additional two days before being examined under a microscope.

All the cells in the wells with sinigrin were dead while there were alot of cells left in the control wells

Figure 2: Microscope comparison of cancer cells in different media. A: RKO cancer cell growing in EMEM+10%FBS. B:RKO cancer cell growing in EMEM+10%FBS + Myrosinase solution after 48h incubation. C:RKO cancer cell growing in EMEM+10%FBS + Myrosinase and Sinigrin solution after 48h incubation

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1023
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Clarke, J., Dashwood, R., & Ho, E. (2008). Multi-targeted prevention of cancer by sulforaphane. Cancer Letters, 269(2), 291-304. doi: 10.1016/j.canlet.2008.04.018