Part:BBa_K3789012:Design
Endochitinase enzyme secretion system
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1023
Illegal PstI site found at 1325
Illegal PstI site found at 1698
Illegal PstI site found at 1800 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1023
Illegal PstI site found at 1325
Illegal PstI site found at 1698
Illegal PstI site found at 1800 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 188
Illegal BamHI site found at 2009
Illegal BamHI site found at 2609 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1023
Illegal PstI site found at 1325
Illegal PstI site found at 1698
Illegal PstI site found at 1800 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1023
Illegal PstI site found at 1325
Illegal PstI site found at 1698
Illegal PstI site found at 1800
Illegal AgeI site found at 2027 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 406
Illegal SapI site found at 2252
Illegal SapI.rc site found at 1362
Design Notes
The endochitinase will degrade the chitins in the yeast cell wall, leading to the yeast death. We need to control this process so it does not happen too early. TPS1 promoter can promote the coding gene expression efficiently with ethanol exits. It enhanced as ethanol concentration increased from 3% to 8%, which is suitable for the time-point (at the end of beer production) we want it to be expressed and secreted.Since the endochitinase needs to be released in the brewing environment to be effective, we choose the common secretion signal sequence “mating factor alpha prepro-leader (MF alpha)” to make the enzyme secretions become possible. Due to these reasons, the system can produce a secretable endochitinase at the end of beer production, which will not split the yeast too early.
Source
TPS1 promoter and Mating factor alpha prepro-leader (MF alpha) come from Saccharomyces cerevisiae S288c.The chit42 coding sequence comes from Trichoderma harziana (CBS226.95). HA-tag sequence comes from the human influenza virus.