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Revision as of 12:59, 26 October 2020
Intro
To lay out what experiments we had to do, we first had to think of the pitfalls of each of the modules, and what could go wrong in different scenarios. Therefore, we chose the following questions to guide our choice of experiments:
- ✧ Will the receptors and accessory proteins have the right subcellular localization?
- ✧ Is the TMD-TF accessory protein able to be cleaved by a TEV protease?
- ✧ Is the TMD-TF accessory protein able to be cleaved by a split TEV protease upon complementation?
- ✧ Will the split-TEV protease be able to cleave the Gα protein and turn on signaling?
- ✧ Are the extracellular domains functioning as intended? Are they dissociated in the absence of the biomarker and will they associate in the presence of the biomarker?
- ✧ How can we optimize the activity of the TEV protease?
And with this in mind, we could start working on all of the different yeast strains we'd need for this!
Cloning
Yeast strains
Assembly nr. |
Description |
X3A |
Ass2A |
Ass2B |
Ass2C |
X3C |
Status |
1 |
IL-10 biosensor using IL-10RI and IL-10II and the split ubiquitin |
Il-10RI-Cub (15) |
empty |
empty |
Il-10RII-Nub (17) |
LexAop-nanoluc |
Confirmed |
2 |
IL-6 biosensor using the IL-6R and truncated sgp130 and split ubiquitin design |
sgp130(D1-D3)-Cub (7) |
empty |
empty |
sIL-6R-Nub (9) |
LexAop-nanoluc |
Confirmed |
3 |
IL-1 biosensor using IL-1R2 and IL-1RAcP and the split ubiquitin design |
sIL-1R2-Cub (11) |
empty |
empty |
sIL-1RAcP-Nub (13) |
LexAop-nanoluc |
Confirmed |
4 |
IL-6 biosensor using the IL-6R and truncated sgp130 and the split-TEV and TMD-LexA-VP16 design |
sgp130(D1-D3)-CTEV (24) |
TMD-LexA-VP16 (44) |
empty |
sIL-6R-NTEV (25) |
LexAop-nanoluc |
no |
5 |
IL-1 biosensor using IL-1R2 and IL-1RAcP and the split-TEV and TMD-LexA-VP16 design |
sIL-1R2-CTEV (28) |
TMD-LexA-VP16 (44) |
empty |
sIL-1RAcP-NTEV (29) |
LexAop-nanoluc |
Confirmed |
6 |
IL-10 biosensor using IL-10RI and IL-10II and the split-TEV and TMD-LexA-VP16 design |
sIL-10RI-CTEV (32) |
TMD-LexA-VP16 (44) |
empty |
sIL-10RII-NTEV (33) |
LexAop-nanoluc |
no |
7 |
IL-6 biosensor using the IL-6R and truncated sgp130 and the split-TEV protease and Galpha cleavage design |
sgp130(D1-D3)-CTEV (24) |
GPA1-mut2 |
LexA-Ste12 |
sIL-6R-NTEV (25) |
LexAop-nanoluc |
no |
8 |
IL-1 biosensor using IL-1R2 and IL-1RAcP and the split-TEV protease and Galpha cleavage design |
sIL-1R2-CTEV (28) |
GPA1-mut2 |
LexA-Ste12 |
sIL-1RAcP-NTEV (29) |
LexAop-nanoluc |
no |
9 |
IL-10 biosensor using IL-10RI and IL-10II and the split-TEV protease and Galpha cleavage design |
sIL-10RI-CTEV (32) |
GPA1-mut2 |
LexA-Ste12 |
sIL-10RII-NTEV (33) |
LexAop-nanoluc |
no |
16 |
IL-6 biosensor using sgp130 and secreted sIL-6R and split ubiquitin design |
sgp130(D1-D6)-cub (1) |
empty |
sIL-6R (5) (ass2B) |
sgp130(D1-D6)-Nub (3) |
LexAop-nanoluc |
Confirmed |
17 |
IL-6 biosensor using sgp130 and secreted sIL-6R and the split-TEV and TMD-LexA-VP16 design |
sgp130(D1-D6)-cTEV (20) |
TMD-TF (44) |
sIL-6R (5) (ass2B) |
sgp130(D1-D6)-nTEV (21) |
LexAop-nanoluc |
no |
18 |
IL-6 biosensor using sgp130 and secreted sIL-6R and the split-TEV protease and Galpha cleavage design |
sgp130(D1-D6)-cTEV (20) and sgp130(D1-D6)-nTEV (21) (57) |
GPA1 mut |
LexA-Ste12 |
sIL-6R (5) |
LexAop-nanoluc |
no |
Assembly nr. |
Description |
X3A |
Ass2A |
Ass2B |
Ass2C |
X3C |
Status |
19 |
For succellular localization assay of sgp130(D1-D6)-Cub |
sgp130(D1-D6)-Cub-sfGFP (2) |
empty |
empty |
empty |
empty |
Confirmed |
20 |
For succellular localization assay of sgp130(D1-D6)-Nub |
empty |
empty |
empty |
sgp130(D1-D6)-Nub-sfGFP (4) |
empty |
no |
21 |
For succellular localization assay of sgp130(D1-D3)-Cub |
sgp130(D1-D3)-Cub-sfGFP (8) |
empty |
empty |
empty |
empty |
Confirmed |
22 |
For succellular localization assay of sIL-6R-Nub |
empty |
empty |
empty |
sIL-6R-Nub-sfGFP (10) |
empty |
Confirmed |
23 |
For succellular localization assay of sIL-1R2-Cub |
sIL-1R2-Cub-sfGFP (12) |
empty |
empty |
empty |
empty |
Confirmed |
24 |
For succellular localization assay of sIL-1RAcP-Nub |
empty |
empty |
empty |
sIL-1RAcP-Nub-sfGFP (14) |
empty |
no |
25 |
For succellular localization assay of sIL-10R1-Cub |
sIL-10R1-Cub-sfGFP (16) |
empty |
empty |
empty |
empty |
Confirmed |
26 |
For succellular localization assay of sIL-10R2-Nub |
empty |
empty |
empty |
sIL-10R2-Nub-sfGFP (18) |
empty |
Confirmed |
27 |
For succellular localization assay of sgp130(D1-D6)-cTEV |
sgp130(D1-D6)-cTEV-sfGFP (22) |
empty |
empty |
empty |
empty |
Confirmed |
28 |
For succellular localization assay of sgp130(D1-D6)-nTEV |
empty |
empty |
empty |
sgp130(D1-D6)-nTEV-sfGFP (23) |
empty |
Confirmed |
29 |
For succellular localization assay of sgp130(D1-D3)-cTEV |
sgp130(D1-D3)-cTEV-sfGFP (26) |
empty |
empty |
empty |
empty |
no |
30 |
For succellular localizationassay of sIL-6R-nTEV |
empty |
empty |
empty |
sIL-6R-nTEV-sfGFP (27) |
empty |
Confirmed |
31 |
For succellular localization assay of sIL-1R2-cTEV |
sIL-1R2-cTEV-sfGFP (30) |
empty |
empty |
empty |
empty |
no |
32 |
For succellular localization assay of sIL-1RAcP-nTEV |
empty |
empty |
empty |
sIL-1RAcP-nTEV-sfGFP (31) |
empty |
Confirmed |
33 |
For succellular localization assay of sIL-10R1-cTEV |
sIL-10R1-cTEV-sfGFP (34) |
empty |
empty |
empty |
empty |
no |
34 |
For succellular localization assay of sIL-10R2-nTEV |
empty |
empty |
empty |
sIL-10R2-nTEV-sfGFP (35) |
empty |
Confirmed |
35 |
For succellular localization assay of sIL-6R |
empty |
empty |
empty |
sIL-6R-sfGFP (6) |
empty |
no |
36 |
For succellular localization assay of TMD-LexA-VP16 |
empty |
TMD-TF-sfGFP (46) |
empty |
empty |
empty |
no |
Assembly nr. |
Description |
X3A |
Ass2A |
Ass2B |
Ass2C |
X3C |
Status |
12.01 |
A2A biosensor with GPA1-mutation 1 |
A2A (R199A) |
GPA1-mutation 1 |
LexA-Ste12 |
empty |
LexAop-nanoluc |
no |
12.02 |
A2A biosensor with GPA1-mutation 2 |
A2A (R199A) |
GPA1-mutation 2 |
LexA-Ste12 |
empty |
LexAop-nanoluc |
no |
12.03 |
A2A biosensor with GPA1-mutation 3 |
A2A (R199A) |
GPA1-mutation 3 |
LexA-Ste12 |
empty |
LexAop-nanoluc |
no |
12.04 |
A2A biosensor with GPA1-mutation 4 |
A2A (R199A) |
GPA1-mutation 4 |
LexA-Ste12 |
empty |
LexAop-nanoluc |
no |
12.05 |
A2A biosensor with GPA1-mutation 5 |
A2A (R199A) |
GPA1-mutation 5 |
LexA-Ste12 |
empty |
LexAop-nanoluc |
no |
13 |
A2A biosensor |
A2A (R199A) |
GPA1 |
LexA-Ste12 |
empty |
LexAop-nanoluc |
no |
14.01 |
A2A biosensor with GPA1-mutation 1 and inducible TEV protease |
A2A (R199A) |
GPA1-mutation1 |
LexA-Ste12 |
scGal1-TEV |
LexAop-nanoluc |
Confirmed |
14.02 |
A2A biosensor with GPA1-mutation 2 and inducible TEV protease |
A2A (R199A) |
GPA1-mutation2 |
LexA-Ste12 |
scGal1-TEV |
LexAop-nanoluc |
Confirmed |
14.03 |
A2A biosensor with GPA1-mutation 3 and inducible TEV protease |
A2A (R199A) |
GPA1-mutation3 |
LexA-Ste12 |
scGal1-TEV |
LexAop-nanoluc |
Confirmed |
14.04 |
A2A biosensor with GPA1-mutation 4 and inducible TEV protease |
A2A (R199A) |
GPA1-mutation4 |
LexA-Ste12 |
scGal1-TEV |
LexAop-nanoluc |
Confirmed |
14.05 |
A2A biosensor with GPA1-mutation 5 and inducible TEV protease |
A2A (R199A) |
GPA1-mutation5 |
LexA-Ste12 |
scGal1-TEV |
LexAop-nanoluc |
no |
15 |
A2A biosensor and inducible TEV protease |
A2A (R199A) |
GPA1 |
LexA-Ste12 |
scGal1-TEV |
LexAop-nanoluc |
Confirmed |
Assembly nr. |
Description |
X3A |
Ass2A |
Ass2B |
Ass2C |
X3C |
Status |
10 |
TMD-LexA-VP16 cleaved by inducible TEV |
empty |
TMD-LexA-VP16 (44) |
empty |
scGal1-TEV |
LexAop-nanoluc |
Confirmed |
37 |
Leucinezippers (K3 and E3) with partially optimised cTEV as in Bba_K1159102 |
(36.1) |
TMD-LexA-VP16 (44) |
empty |
(37.1) |
LexAop-nanoluc |
no |
38 |
Leucinezippers (K3 and E3) with most optiimsed cTEV |
(38.1) |
TMD-LexA-VP16 (44) |
empty |
(37.1) |
LexAop-nanoluc |
no |
39 |
Leucinezippers (K3 and E3) with Wt cTEV as in Bba_K1965020 |
(39.1) |
TMD-LexA-VP16 (44) |
empty |
(37.1) |
LexAop-nanoluc |
no |
40 |
Leucinezippers (ZR and ZE) with partially optimised cTEV as in Bba_K1159102 |
(36.2) |
TMD-LexA-VP16 (44) |
empty |
(37.2) |
LexAop-nanoluc |
no |
41 |
Leucinezippers (ZR and ZE) with optimised cTEV |
(38.2) |
TMD-LexA-VP16 (44) |
empty |
(37.2) |
LexAop-nanoluc |
no |
42 |
Leucinezippers (ZR and ZE) with Wt cTEV as in Bba_K1965020 |
(39.2) |
TMD-LexA-VP16 (44) |
empty |
(37.2) |
LexAop-nanoluc |
no |
Other strains:
Assembly nr. |
Description |
X3A |
Ass2A |
Ass2B |
Ass2C |
X3C |
Status |
11 |
control for reporter leakage |
empty |
empty |
empty |
empty |
LexAop-nanoluc |
no |
localization assays
sgp130(D1-D3)-Cub-sfGFP - Picture left: Analysis similar to picture on the right. Inclusion bodies are visible on the brightfield image, and slightly more protein localizes to the plasma membrane. Picture right: Faint localization of sgp130(D1-D3)-Cub in the endoplasmatic reticulum, with some protein also localizing to the plasma membrane. The majority of the protein localized to inclusion bodies/vacuoles.
sIL-6R-Nub-sfGFP - Picture left: The majority of sIL-6R-Nub-sfGFP localizes to inclusion bodies near the plasma membrane of Saccharomyces cerevisiae cells. Little to none of the protein can be found localized to other subcellular compartments. Picture right: sIL-6R-Nub-sfGFP can be observed in both smaller and larger inclusion bodies, similar to the picture on the left. Faint localization uniformly in the cytosol and plasma membrane.
sIL-10R1-Cub-sfGFP - Picture left: sIL-10R1-Cub-sfGFP is shown to primarily localize to the plasma membrane of S. cerevisiae cells and the endoplasmatic reticulum Picture right: Analysis similar to picture on the left but more faint staining of the endoplasmatic reticulum. Small inclusion bodies can be observed near the plasma membrane.
sIL-10R2-Nub-sfGFP - Picture left: sIL-10R2-Nub-sfGFP shows localization within the entirety of the S. cerevisiae cell and the plasma membrane without distinction of which subcellular compartments that the protein resides in. Picture right: Analysis mostly similar to picture on the left. The picture indicates less localization of the protein in the plasma membrane compared to the cytosolic and nuclear compartment, however, for the two large cells that can be observed.
sIL-6R-nTEV-sfGFP - Picture left: sIL-6R-nTEV-sfGFP can be observed to localize primarily to the plasma membrane, both in small and medium-sized inclusion bodies, as well as in small amount of “free” form. Some protein localizes to the cytosolic compartment as well, seemingly also in inclusion bodies or in the endoplasmatic reticulum. Picture right: sIL-6R-nTEV-sfGFP clearly observed to localize primarily to the plasma membrane of the larger of the two cells with fluorescence being strongest in this compartment, whereas small inclusion bodies can be observed in the smaller of the two cells.
sIL-1RAcP-nTEV-sfGFP - Picture left: sIL-1RAcP-nTEV-sfGFP localizes uniformly throughout the cytosol of the cell in what looks like inclusion bodies, seemingly without localizing, or localizing very little, to the plasma membrane. Picture right: Uniform localization of sIL-1RAcP-nTEV-sfGFP throughout the cytosol of the cells and near the plasma membrane, albeit not in inclusion bodies as seen in the picture on the left.
sIL-10R2-nTEV-sfGFP - sIL-10R2-nTEV-sfGFP localizes primarily to inclusion bodies near the plasma membrane of the S. cerevisiae cells. This is particularly evident in the top right and bottom cells. However, much of the protein also localizes within the cytosol. While unclear, it may seem that some of the protein localizes within the endoplasmatic reticulum.
Cleavage of membrane bound transcription factor