Difference between revisions of "Team:Slovenia/Parts"
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<p>We improved the BioBrick <a href="http://parts.igem.org/Part:BBa_K737005:Experience">BBa_K737005</a>, deposited by team OUC-China in 2012, coding for the | <p>We improved the BioBrick <a href="http://parts.igem.org/Part:BBa_K737005:Experience">BBa_K737005</a>, 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 | 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 | ||
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Revision as of 00:03, 19 October 2016
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 this year 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. The parts can be divided into 4 collections based on their function:
- the collection of mechano- and ultrasound-sensing parts,
- the collection of orthogonal split and whole proteases with their reporters,
- the collection of parts for logic functions and
- the collection of parts for protease-triggered protein secretion.
List of parts
| Construct | Biobrick number | |
|---|---|---|
| MscS:HA | BBa_K1965000 | |
| P3:FAStm:HA:TRPC1:Myc | BBa_K1965002 | |
| FLAG:GvpC | BBa_K1965003 | |
| Au1:GvpA | BBa_K1965004 | |
| P3:cLuc | BBa_K1965005 | |
| nLuc:AP4 | BBa_K1965006 | |
| fLUC:TEVs | BBa_K1965010 | |
| nLuc:M13 | BBa_K1965014 | |
| CaM(E104Q):cLuc | BBa_K1965015 | |
| His:CRY:Myc:nTEV | BBa_K1965019 | |
| His:CIBN:cTEV:HA | BBa_K1965020 | |
| nLuc:AP4:TEVs:P3mS | BBa_K1965021 | |
| nLuc:AP4:TEVs:P3mS-2A | BBa_K1965022 | |
| PPVp | BBa_K1965025 | |
| FKBP:cPPVp | BBa_K1965026 | |
| FRB:nPPVp | BBa_K1965027 | |
| ss:TagRFP:AU1:furS:TM:3xTEVs:KKMP | BBa_K1965030 | |
| cycLuc_SbMVs | BBa_K1965031 | |
| FKBP:cSbMVp:HA | BBa_K1965032 | |
| Myc:FRB:nSbMVp | BBa_K1965033 | |
| cycLuc_SuMMVs | BBa_K1965034 | |
| FKBP:SuMMVp:HA | BBa_K1965035 | |
| Myc:FRB:SuMMVp | BBa_K1965036 | |
| cycLuc_TEVs | BBa_K1965037 | |
| SbMVp | BBa_K1965040 | |
| SuMMVp | BBa_K1965041 | |
| 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 BioBrick BBa_K737005, 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.
