Team:UCopenhagen/Results

Intro

As explained on our project design page, our design is highly modular, with four different pairs of extracellular receptor domains, as well as three different intracellular designs for signal transduction. This creates a lot of room to switch different modules around, but at the same time, it also creates multiple points for optimization and verification. In order to verify that all of our different modules work, it meant that we'd have to break down our designs into all of their smaller constituents, and create experiments around them in order to test their functionality. This, of course, had to happen in parallel with creating and assaying our complete whole cell biosensors.

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

First we had to create the vectors, that we wanted to use in our experiments. Five different vector backbones were used for itegrating our genes into the yeast genome. Insertion of our genes and their promoters was achieved using USER cloning and the Escterichia coli strain mach1 as our platform. Our cloning framework consisted of the following steps:

Figure 1: cloning flowchart Obtain relevant genes and promoters → PCR amplification → USER ligation → transform E. coli → colony PCR → sequencing → transform yeast → yeast colony PCR

Most of the gene fragments we have used, we ordered as synthetic DNA from IDT and Twist bioscience after designing and codon optimizing them for expression in yeast using Benchling. Other fragments like the promoters we used and some of the genes, e.g. our reporter, were already available in our supervisor Sorterios Kampranis' lab

When PCR amplifying the gene fragments, we used primers carrying USER overhangs. In the USER ligation (maybe link to or pop-up: explanation of USER cloning) the PCR products are digested with USER enzyme mix. This creates 3’ overhangs that allow the fragments to be seamlessly ligated to each other and to a backbone vector in a directional manner. To confirm that the gene fragments were correctly amplified, the PCR products were analyzed using gel electrophoresis on an agarose gel.

Figure 2: Picture of an agarose gel after gel electrophoresis of some of our PCR fragments. The bands that indicate the length of our desired PCR products ar marked with a star (*).

Some PCR reactions gave us multiple bands. Instead of PCR clean-up, a gel extraction was performed in order to obtain the desired fragments. The fragments were then used in USER ligations as described above. Subsequently competent E. coli cells were transformed with the products of the USER ligation and grown on agar plates with carbenicillin O/N. This allowed for selection for cells that had been transformed with plasmids from our USER ligation since all our plasmid backbones contained a gene encoding β-lactamase.

Figure 3: Stock image of plate. remember to find a pic of our plates.

To identify colonies with plasmids where the desired constructs had been inserted into the vector backbone, we picked between 4-16 colonies for colony PCR. We used primers that annealed to the vector backbone on either side of the USER cassette. If nothing was inserted, analyzing the PCR product with gel electrophoresis would yield a band at 300 bp, a band at the sum of our gene and promoter + 300 bp would mean that the construct had been successfully inserted in the backbone vector. (consider making this a picture text) By using primers that anneal to the backbone we automatically had a process control since no bands would mean that something had gone wrong with the colony PCR reaction.

Figure 4a: A map of one of our vector constructs. The vector consists of the Ass2c backbone with a gene encoding TEV protease and an inducible promoter, scGAL1, inserted. Primers used in the colony PCR are shown. The distance between the forward and backward primers is about 1700bp. Bands of approximately this size would indicate that the plasmid had our full construct inserted.

Gel electrophoresis of colony PCR product. Green arrow: The band indicated that the colony had been transformed with the full construct from figure 4a. Red arrow: Colony with an empty plasmid. Yellow arrow: The plasmid did not have our full construct inserted. It might be caused by a deletion or a single missing fragment.

Colonies with our construct inserted were inoculated overnight. After plasmid purification, the vectors were sent for sequencing to confirm that our construct had been inserted without mutations. In total, we managed to clone 53 constructs. The following table shows what plasmids we created or planned to create:

After confirming the right sequence, the constructs could be used for creating different yeast strains.

Yeast strains

To create the strains we needed for testing our system, we applied the 5-assembly system for stable transfection into the chromosome X site 3 locus using homologous recombination. Insertion of vectors into the locus requires all five vectors of the assembly system to be present and integrated.

Figure 5: The 5 assembly system

Correct insertion of all five vectors can thus be confirmed with a single colony PCR with three primers: a forward and a reverse primer anneal up- and down-stream of the homologous recombination site respectively and a second forward primer anneals to the X3C backbone.

Figure 5: Yeast colony PCR

A band at about 1500 bp would mean no insertion into the locus, while a band at 1000 bp implies that all five vectors have been inserted successfully. In total 22 different strains were created. Beneath are tables showing all the strains we created or intended to create in order to do the different experiments:

Biosensors

Our biosensors

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

Localization assays

These strains were designed in order to do subcellular localization assays on the different components of our biosensors and some of the accessory proteins needed for the designs to work.

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

Characterizing modified Gαs

For our third design to work, we needed to modify the endogenous g-protein subunit α, GPA1, from S. cerevisiae. These are the strains we needed to test the functionality of the different modified GPA1 proteins we designed.

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

Strains for other experiments

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

Design 1

In this design, the extracellular IL-receptors were fused with either a C- or N-terminal part of a split-ubiquitin protein. Upon extracelluar association in the presence of IL molecules, the two halves will reconstitute to ubiquitin and be recognized by deubiqitinase, resulting in the release of a LexA transcription factor.

Design 2

In this design, amplification is achieved by adding multiple membrane bound transcription factors. Here, the extracellular IL-receptors were fused with either a C- or N-terminal part of a split-TEV protease. Upon extracelluar association in the presence of IL molecules, the two halves will reconstitute to a functioning TEV protease. This protease recognizes our membrane bound transcription factor LexA and releases it from the membrane.

Design 3

In this design, amplification is achieved by hijacking the pheromone pathway through a modified G-alpha subunit. The extracellular IL-receptors were fused with a split-TEV protease that can cleave the G-alpha subunit. Upon extracelluar association the two halves will reconstitute and cleave G-alpha, releasing the beta-gamma subunit that transduces the signal.

localization assays

Confocal microscopy was used to validate the presence of our proteins of interest within their respective subcellular compartments, or within the plasma membrane of Saccharomyces cerevisiae cells. Superfolding green fluorescent protein was linked to the C-terminal of the protein product of the biobrick for the visualization purpose. All pictures were taken with a 150 μm pinhole. Use the slider below for easy differentiation between fluorescent microscopy and brightfield microscopy!

flourescence brightfield

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

For the intermediate biosensor design where a split TEV protease releases transcription factors from the membrane we needed to create a transformembrane protein,k that has a transcripction factor on the intracellular side. Thus we designed an accessory protein with a transcription factor linked to a transmembrane domain through a flexible linker that had a cut site for the TEV protease. This experiment was designed in order to verify that a TEV protease could induce signalling by cleaving the flexible linker and releasing the transcription factor. For the experiment we integrated the accessory protein, an inducible TEV protease and a nanoLuc luciferase into S. cerevisiae as described above (see assembly 10 in the table). The strain was grown to an OD600=5,1 and incubated at 30°C for 17 hours in either glucose or galactose + raffinose media to repress or activate the transcription of the TEV protease respectively. Next, a luciferase assay was carried out on the strain