All parts

Sonicell project introduces rapid cellular response to a combination of external stimuli like light, ultrasound or small molecules. Our system incorporates a module for the enhanced sensitivity to ultrasound or other mechanical stimuli sensed by a calcium-dependent reporter. Furthermore we designed a module that can combine several input signals in a novel signaling pathway/logic professing based on the collection of (split) orthogonal proteases. Those proteases can also cleave the endoplasmic reticulum retention peptide segment, which results in a rapid secretion of proteins or peptides.

Our BioBrick submission, which consists of 52 parts, therefore represents a collection that covers the broad range the aforementioned tasks aimed to function in mammalian cells, however many should also function in other types of a chassis. All the favorite parts and a large majority of the rest were successfully tested in mammal cells. The parts can be divided into 4 collections based on their function:

 List of parts

Favorites Construct BioBrick number
newAtiGEM MscS:HA BBa_K1965000
newAtiGEM P3:FAStm:HA:TRPC1:Myc BBa_K1965002
newAtiGEM FLAG:GvpC BBa_K1965003
newAtiGEM Au1:GvpA BBa_K1965004
newAtiGEM P3:cLuc BBa_K1965005
newAtiGEM nLuc:AP4 BBa_K1965006
newAtiGEM fLUC:TEVs BBa_K1965010
newAtiGEM nLuc:M13 BBa_K1965014
newAtiGEM CaM(E104Q):cLuc BBa_K1965015
newAtiGEM His:CRY:Myc:nTEV BBa_K1965019
newAtiGEM His:CIBN:cTEV:HA BBa_K1965020
newAtiGEM nLuc:AP4:TEVs:P3mS BBa_K1965021
newAtiGEM nLuc:AP4:TEVs:P3mS-2A BBa_K1965022
newAtiGEM PPVp BBa_K1965025
newAtiGEM FKBP:cPPVp BBa_K1965026
newAtiGEM FRB:nPPVp BBa_K1965027
newAtiGEM ss:TagRFP:AU1:furS:TM:3xTEVs:KKMP BBa_K1965030
newAtiGEM cycLuc_SbMVs BBa_K1965031
newAtiGEM FKBP:cSbMVp:HA BBa_K1965032
newAtiGEM Myc:FRB:nSbMVp BBa_K1965033
newAtiGEM cycLuc_SuMMVs BBa_K1965034
newAtiGEM FKBP:SuMMVp:HA BBa_K1965035
newAtiGEM Myc:FRB:SuMMVp BBa_K1965036
newAtiGEM cycLuc_TEVs BBa_K1965037
newAtiGEM SbMVp BBa_K1965040
newAtiGEM SuMMVp BBa_K1965041
newAtiGEM cycLuc_PPVs BBa_K1965042
TRPC1:Myc BBa_K1965001
His:CRY:Myc:nLuc BBa_K1965007
His:CIBN:cLuc:HA BBa_K1965008
Myc:TEVP:HA BBa_K1965009
P5:cLuc BBa_K1965011
P7:cLuc BBa_K1965012
P9:cLuc BBa_K1965013
nTEV:M13 BBa_K1965016
CaM(E104Q):Ctev BBa_K1965017
CaM(E31Q,E104Q):cLuc BBa_K1965018
Myc:nLuc:GS10:AP6 BBa_K1965023
erTEVp BBa_K1965024
ss:TagRFP:AU1:TEVs:KDEL BBa_K1965028
ss:TagRFP:AU1:furS:TD1:TEVs BBa_K1965029
FKBP:cTEV BBa_K1965038
FRB:nTEVp BBa_K1965039
CRY:nPPV BBa_K1965043
CIB:cPPV BBa_K1965044
cLuc:A:HA BBa_K1965045
Myc:B:nLuc BBa_K1965046
cLuc:A:PPVs:B'2a:HA BBa_K1965047
Myc:A':TEVs:B:nLuc BBa_K1965048
P3:GS6:PPVs:GS6:cLuc:HA BBa_K1965049
P5:GS6:PPVs:GS6:cLuc:HA BBa_K1965050
Myc:nLuc:GS6:TEVs:GS6:AP4 BBa_K1965051

 Part improvements

We improved the BioBricks BBa_K737005 and BBa_K737017, deposited by team OUC-China in 2012, coding for the GvpC protein. This protein is one of two proteins that compose protein gas vesicles, which were used in our project to enhance the sensitivity of mammalian cells to mechanical stimuli. We improved the part by adding a FLAG tag, which allowed us to analyze the expression of the part form the cells via western blot and to observe the subcellular localization of the protein by confocal microscopy. We further characterized the part by expressing it in mammalian cells together with GvpA, showing that these proteins substantially increase the cell sensitivity to ultrasound. This part also plays a central role in our Mechanosensing collection.

We also improved BBa_K157010, a transmembrane domain used by 2008 iGEM Freiburg team. We used this domain as an anchor to which we attached the TagRFP protein on the N-terminal side and the ER retention signal on the C-terminal side. By western blots and confocal fluorescent microcopy we were able to show that this part can not only be used for plasma membrane localization but can also be retained in the ER with the simple addition of a 4-amino-acid retention signal KKMP. Upon induced proteolytic cleavage of the retention signal by inducible split protease, the protein was translocated to the trans GA and the plasma membrane, where TagRFP was cleaved by furin and secreted into the medium.