Team:Slovenia/Part Collection
Part Collections
- 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.
Mechanosensing Collection
Our BioBricks can work together as a coordinated system, but can also be divided into four smaller collections as mentioned above.
Our Mechanosensing Collection is composed of mechanosensitive ion channels and coding parts for protein gas vesicles, which enhance the sensitivity of mammalian cells to ultrasound or other mechanical stimuli, as well as a calcium influx measurement device based on the formation of a complex between calmodulin and M13 peptide with split luciferase, which results in the luminescence upon calcium influx.
| Construct | Biobrick number |
|---|---|
| MscS:HA | BBa_K1965000 |
| TRPC1:Myc | BBa_K1965001 |
| P3:FAStm:HA:TRPC1:Myc | BBa_K1965002 |
| FLAG:GvpC | BBa_K1965003 |
| Au1:GvpA | BBa_K1965004 |
| nLuc:M13 | BBa_K1965014 |
| CaM(E104Q):cLuc | BBa_K1965015 |
| nTEV:M13 | BBa_K1965016 |
| CaM(E104Q):Ctev | BBa_K1965017 |
| CaM(E31Q,E104Q):cLuc | BBa_K1965018 |
Touchpaint Collection
A Touchpaint Collection is a subset of the Mechanosensing Collection that enables to convert mechanical stimulus of mammalian cells into light. This set contains the parts to enhance the sensitivity of cells to mechanical stimulus and the luciferase reporter. While this collection has been used to draw on cells it has many other uses, such as detection of the shear flow, ultrasound and other types of direct and indirect mechanical stress.
| Construct | Biobrick number |
|---|---|
| MscS:HA | BBa_K1965000 |
| FLAG:GvpC | BBa_K1965003 |
| Au1:GvpA | BBa_K1965004 |
| nLuc:M13 | BBa_K1965014 |
| CaM(E104Q):cLuc | BBa_K1965015 |
Orthogonal Protease Collection
Our Orthogonal Protease Collection (nominated for Best Collection) is composed of a set of site-specific proteases that recognize different 7-aminoacid residue motif targets (PPVp, SuMMVp, SbMVp and TEVp). The proteases were tested and are deposited as single chain coding sequences and as split protein fragments for light- and chemically-inducible reconstitution. The collection also contains the corresponding cyclic luciferase reporters for each of the proteases. We determined that the new proteases are even more active than the TEVp that has been the standard in biotechnology for several decades. We demonstrated that the split versions of all four proteases can be activated by different external stimuli, such as small molecules or light, demonstrating the robustness and versatility of the system. These proteases have been used to design signaling pathways and logic circuits in mammalian cells operating at the post-translational level.
| Construct | Biobrick number |
|---|---|
| His:CRY:Myc:nLuc | BBa_K1965007 |
| His:CIBN:cLuc:HA | BBa_K1965008 |
| Myc:TEVP:HA | BBa_K1965009 |
| fLUC:TEVs | BBa_K1965010 |
| His:CRY:Myc:nTEV | BBa_K1965019 |
| His:CIBN:cTEV:HA | BBa_K1965020 |
| erTEVp | BBa_K1965024 |
| PPVp | BBa_K1965025 |
| FKBP:cPPVp | BBa_K1965026 |
| FRB:nPPVp | BBa_K1965027 |
| 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 |
| FKBP:cTEV | BBa_K1965038 |
| FRB:nTEVp | BBa_K1965039 |
| SbMVp | BBa_K1965040 |
| SuMMVp | BBa_K1965041 |
| cycLuc_PPVs | BBa_K1965042 |
| CRY:nPPV | BBa_K1965043 |
| CIB:cPPV | BBa_K1965044 |
Protease-based Logic Collection
Our Protease-based Logic Collection is composed of sets of parallel, antiparallel and destabilized coiled coils in fusion with parts of split luciferase. These constructs represent a tool for constructing logic circuits in mammalian cells operating at the post-translational level. The input signals for our logic operations is proteolytic activity of selected orthogonal proteases, representing constitutive logic gates, or split TEV and PPV proteases on inducible systems, representing inducible logic gates.
| Construct | Biobrick number |
|---|---|
| P3:cLuc | BBa_K1965005 |
| nLuc:AP4 | BBa_K1965006 |
| P5:GS10:cLuc:HA | BBa_K1965011 |
| P7:GS10:cLuc:HA | BBa_K1965012 |
| P9:GS10:cLuc:HA | BBa_K1965013 |
| nLuc:AP4:TEVs:P3mS | BBa_K1965021 |
| nLuc:AP4:TEVs:P3mS-2A | BBa_K1965022 |
| Myc:nLuc:GS10:AP6 | BBa_K1965023 |
| 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 |
FastER Secretion Collection
Our FastER Secretion Collection is composed of parts coding for the TagRFP reporter with different ER retention signals, attached to the reporter via a TEV cleavage site. In this way, a protein can aggregate in the ER and be secreted upon induction by proteolytic cleavage with one of our inducible split TEV proteases without having to wait for the transcription and translation of the protein.
| Construct | Biobrick number |
|---|---|
| ss:TagRFP:AU1:TEVs:KDEL | BBa_K1965028 |
| ss-TagRFP:AU1:furS:TM:TEVs | BBa_K1965029 |
| ss:TagRFPA:U1:furS:TM:3xTEVs:KKMP | BBa_K1965030 |
