Team:UCopenhagen/Results



Introduction

The ambition of CIDosis is to design and create an inflammation-detecting patch, containing a Saccharomyces cerevisiae-based interleukin biosensor able to track concentrations of selected interleukins in sweat. For this, we decided to create three biosensor designs with progressing complexity based on interleukin receptor association. The general CIDosis biosensor design (project design page) is highly modular as exemplified here with four different biosensors (IL-1, IL-10, IL-6 design1 IL-6 design 2) each consisting of a different extracellular receptor domain pair. Each biosensor can be constructed with either of three different intracellular signal transduction modules.

The modular nature of the biosensor enables multiple setups for different purposes. From the implementation point of view, it also creates multiple points for optimization and verification. In order to verify that all of our different modules work we divided designs into smaller constituents and created experiments around them in order to test their functionality. This was done 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:
Figure 1: General biosensor design.

  • ✧ Are the extracellular domains functioning as intended? Are they dissociated in the absence of the interleukins and will they associate in the presence of the interleukins?
  • ✧ Do the receptors and accessory proteins have the intended subcellular localization?
  • ✧ Is the membrane bound transcription factor of the intermediate design able to be cleaved by a TEV protease?
  • ✧ Is the TEV protease able to cleave the modified Gα (Gpa1p) proteins and trigger the yeast pheromone pathway?
  • ✧ Do the modified Gα (Gpa1p) proteins retain their ability to transduce signalling from a GPCR?

To answer these questions we followed the scientific workflow shown in figure 2.

Figure 2: Scientific workflow - Obtaining synthetic genes and promoters → PCR amplification → USER plasmid ligation → transformation into Escherichia coli → colony PCR → sequencing → yeast transformation → yeast colony PCR → Biosensor (and other) assays.

Cloning

First we had to create the plasmids that we wanted to use in our experiments. All cloning steps were performed with USER cloning. For an explanation of the concepts behind USER cloning, watch our introductive video on the education page. For further details, see also the article by Geu-Flores et al. 2007.
Most of the gene fragments used were ordered as synthetic DNA from IDT and Twist Bioscience after designing and codon optimizing them for expression in S. cerevisiae 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
As a first step, we started by PCR amplifying the promoters and ORFs needed for biosensor construction. To confirm that the gene fragments were correctly amplified, the PCR products were analyzed using gel electrophoresis on an agarose gel.
Figure 3: Representative image of a gel electrophoresis of positive PCR fragment amplifications. The bands that indicate the length of our desired PCR products are marked with a star (*).
The PCR products with the correct size were isolated by gel extraction.

Then, the fragments were digested with the USER enzyme to create custom sticky ends. These fragments were ligated by incubation with the relevant linearized vector. Subsequently, competent E. coli cells were transformed with the products of the USER ligation and grown on agar plates with carbenicillin. 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 (AmpR).
Figure 4: Representative picture of plates with transformed bacteria and yeast. Background: Our transformed E.coli was grown on agar plates with carbinicilin to select for colonies containing our plasmids. Foreground: Transformed S. cerevisiae was grown on glucose plates without uracil to select for the colonies with the URA3 gene in the X3A vector inserted. The vectors used for yeast transformation are described in the next chapter.

To identify colonies with plasmids with the desired inserts, 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 (figure 5a). If nothing was inserted, analyzing the PCR product with gel electrophoresis would yield a band at 300 bp. A band at the sum of the length of our gene and promoter + 300 bp from flanking regions would mean that the construct had been successfully inserted in the backbone vector (figure 5b). 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 5b: Gel electrophoresis of colony PCR products. Green arrow: The band indicated that the colony had been transformed with the full construct from figure 5a. 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.
Figure 5a: 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.

Colonies corresponding to positive colony PCRs were grown overnight in liquid media with antibiotic selection and plasmid purified with a miniprep kit. The resulting plasmids 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.
Constructs
SP= Wsc1 ER import signal peptide, TMD= Wsc1 transmembrane domain.

Construct name

Vector

Promoter

Domains in gene

Status

Sgp130(D1-D6)-Cub

X3A

pCCW12

SP-sgp130(D1-D6)-TMD-Cub-LexA-VP16

Done

Sgp130(D1-D6)-Cub

X3A

pCCW12

SP-sgp130(D1-D6)-TMD-Cub-LexA-VP16-sfGFP

Done

Sgp130(D1-D6)-Nub

Ass2C

pTDH3

SP-Sgp130(D1-D6)-TMD-Nub

Done

Sgp130(D1-D6)-Nub

Ass2C

pTDH3

SP-Sgp130(D1-D6)-TMD-Nub-sfGFP

No - two colonies had mutations did not sequence third

sIL-6R (secreted)

Ass2C

pPGK1

Alpha factor-pre-pro-sequence -sIL-6R

Done

sIL-6R (secreted)

Ass2B

pPGK1

Alpha factor-pre-pro-sequence -sIL-6R

Done

sIL-6R (secreted)

Ass2C

pPGK1

Alpha factor-pre-pro-sequence -sIL-6R

Done

Sgp-130(D1-D3)-Cub

X3A

pCCW12

SP-Sgp130(D1-D3)-TMD-Cub-LexA-VP16

Done

Sgp-130(D1-D3)-Cub

X3A

pCCW12

SP-Sgp130(D1-D3)-TMD-Cub-LexA-VP16-sfGFP

Done

sIL-6R-Nub

Ass2C

pTDH3

SP-sIL-6R-TMD-Nub

Done

sIL-6R-Nub

Ass2C

pTDH3

SP-sIL-6R-TMD-Nub-sfGFP

Done

sIL-1R2-Cub

X3A

pCCW12

SP-sIL-1R2-TMD-Cub-LexA-VP16

Done

sIL-1R2-Cub

X3A

pCCW12

SP-sIL-1R2-TMD-Cub-LexA-VP16-sfGFP

Done

sIL-1RAcP-Nub

Ass2C

pTDH3

SP-sIL-1RAcP-TMD-Nub

Done

sIL-1RAcP-Nub

Ass2C

pTDH3

SP-sIL-1RAcp-TMD-Nub-sfGFP

Redo USER ligation

sIL-10R1-Cub

X3A

pCCW12

SP-sIL-10R1-TMD-Cub-LexA-VP16

Done

sIL-10R1-Cub

X3A

pCCW12

SP-sIL-10R1-TMD-Cub-LexA-VP16-sfGFP

Done

sIL-10R2-Nub

Ass2C

pTDH3

SP-sIL-10R2-TMD-Nub

Done

sIL-10R2-Nub

Ass2C

pTDH3

SP-sIL-10R2-TMD-Nub-sfGFP

Done

MJDOD

X3C

LexAop

Already in lab

Already in lab

Sgp-130(D1-D6)-CTEV

X3A

pCCW12

SP-Sgp-130(D1-D6)-TMD-CTEV

Done

Sgp-130(D1-D6)-NTEV

Ass2C

pTDH3

SP-Sgp-130(D1-D6)-TMD-NTEV

Done

Sgp-130(D1-D6)-CTEV

X3A

pCCW12

SP- Sgp-130(D1-D6)-TMD-CTEV-sfGFP

Done

Sgp-130(D1-D6)-NTEV

Ass2C

pTDH3

SP- Sgp-130(D1-D6)-TMD-NTEV-sfGFP

Done

Sgp130-(D1-D3)-CTEV

X3A

pCCW12

SP-Sgp130-(D1-D3)-TMD-CTEV

Done

sIL-6R-NTEV

Ass2C

pTDH3

SP-sIL-6R-TMD-NTEV

Done

Sgp130(D1-D3)-CTEV

X3A

pCCW12

SP-sgp130-TMD-CTEV-sfGFP

USER-ligation produced stop codon.
New primer needed.

sIL-6R-NTEV

Ass2C

pTDH3

SP-sIL-6R-TMD-NTEV-sfGFP

Done

sIL-1R2-CTEV

X3A

pCCW12

SP-sIL-1R2-TMD-CTEV

Done

sIL-1RAcp-NTEV

Ass2C

pTDH3

SP-sIL-1RAcp-TMD-NTEV

Done

sIL-1R2-CTEV

X3A

pCCW12

SP-sIL-1R2-TMD-CTEV-sfGFP

USER-ligation produced stop codon. New primer needed

sIL-1RAcP-NTEV

Ass2C

pTDH3

SP-sIL-1RAcP-TMD-NTEV-sfGFP

Done

sIL-10R1-CTEV

X3A

pCCW12

SP-sIL-10R1-TMD-CTEV

Done

sIL-10R2-NTEV

Ass2C

pTDH3

SP-sIL-10R2-TMD-NTEV

Done

sIL-10R1-CTEV

X3A

pCCW12

SP-sIL-10R1-TMD-CTEV-sfGFP

USER-ligation produced stop codon.
New primer needed

sIL-10R2-NTEV

Ass2C

pTDH3

SP-sIL-10R2-TMD-NTEV-sfGFP

Done

E3-CTEV (BBa_K1159102)

X3A

SP-E3-TMD-CTEV(BBa_K1159102)

Do colony PCR

ZE-CTEV (BBa_K1159102)

SP-ZE-TMD-CTEV (BBa_K1159102)

Do colony PCR

K3-NTEV

SP-K3-TMD-NTEV

Do colony PCR

ZR-NTEV

SP-ZR-TMD-NTEV

Do colony PCR

E3-CTEV (truncated C-terminal)

SP-E3-TMD-CTEV(truncated C-terminal)

Do colony PCR

ZE-CTEV (truncated C-terminal)

SP-ZE-TMD-CTEV (truncated C-terminal)

Do colony PCR

E3-CTEV (wt)

SP-E3-TMD-CTEV(wt)

Do colony PCR

ZE-CTEV (wt)

SP-ZE-TMD-CTEV (wt)

Do colony PCR

40

cancelled

41

cancelled

42

cancelled

43

cancelled

44

Ass2A

pPGK1

SP-TMD-ENLYFQG-LexA-VP16

Done

45

cancelled

46

Ass2a

ppgk1

SP-TMD-ENLYFQG-LexA-VP16-sfGFP

Done

47

cancelled

48

new plan for G-alpha

49

new plan for G-alpha

50

new plan for G-alpha

51

Ass2A

pPGK1

already in lab

52

Ass2C

scGAL1

TEV protease

done

53

Ass2B

pRET

Ste12

cancelled

54

Ass2B

pRET

LexA-Ste12

already in lab

55

Ass2B

pFIG

LexA-nanoluciferase

already in lab

56

X3A

pCCW12

A2A (R199A)

already in lab

GPA1 mutations:

GPA1mut1

Ass2A

pPGK1

GPA1 with cut site inserted at site one

done

GPA1mut2

Ass2A

pPGK1

GPA1 with cut site inserted at site two

done

GPA1mut3

Ass2A

pPGK1

GPA1 with cut site inserted at site three

done

GPA1mut4

Ass2A

pPGK1

GPA1 with cut site inserted at site four

done

GPA1mut5

Ass2A

pPGK1

GPA1 with cut site inserted at site five

done

Table 1: Constructs

Yeast strains

All the yeast strains (table 2) were constructed by genomic integration of the different modules into chromosome X site 3 using the homologous recombination based 5-plasmid assembly system (unpublished) available at the host lab (figure 5).
Figure 6: The 5 assembly system. Convenient assembly of different biosensors through the shuffling of parts in the five modules dedicated to different functions is allowed by the modular structure of the 5 assembly system (Unpublished). Extra flexibility is provided by the Aux module by accommodating additional parts, such as for constructing multi-part reporters. One assembles the biosensor by co-transforming five linearized module plasmids that contain a promoter and a coding sequence, with both being flanked by homologous recombination regions. To ensure consistent and controllable expression of the module genes of interest, the five modules are then stably integrated into the genome of Saccharomyces cerevisiae (Unpublished).

Correct insertion of all five vectors can was 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 on the yeast chromosome respectively and a second forward primer anneals to the inserted construct (explained in figure 7).
Figure 7: Yeast colony PCR A) 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. B) Arrow a: Positive band at 1000 bp. Arrow b: Negative band at 1500 bp.


In total, 21 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 

CD1 

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 

CD2 

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 

CD3 

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 

CD4 

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 

CD5 

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 

CD6 

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 

CD7 

IL-6 biosensor using the IL-6R and truncated sgp130 and the split-TEV protease and  cleavage  design 

sgp130(D1-D3)-CTEV (24) 

GPA1-mut2 

LexA-Ste12 

sIL-6R-NTEV (25) 

LexAop-nanoluc 

no 

CD8 

IL-1 biosensor using IL-1R2 and IL-1RAcP and the split-TEV protease and  cleavage  design 

sIL-1R2-CTEV (28) 

GPA1-mut2 

LexA-Ste12 

sIL-1RAcP-NTEV (29) 

LexAop-nanoluc 

no 

CD9 

IL-10 biosensor using IL-10RI and IL-10II and the split-TEV protease and  cleavage design 

sIL-10RI-CTEV (32) 

GPA1-mut2 

LexA-Ste12 

sIL-10RII-NTEV (33) 

LexAop-nanoluc 

no 

CD16 

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 

CD17 

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 

CD18 

IL-6 biosensor using sgp130 and secreted sIL-6R and the split-TEV protease and  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 

CD19 

For succellular localization assay of sgp130(D1-D6)-Cub 

sgp130(D1-D6)-Cub-sfGFP (2) 

empty 

empty 

empty 

empty 

Confirmed 

CD20 

For succellular localization assay of sgp130(D1-D6)-Nub 

empty 

empty 

empty 

sgp130(D1-D6)-Nub-sfGFP (4) 

empty 

no 

CD21 

For succellular localization assay of sgp130(D1-D3)-Cub 

sgp130(D1-D3)-Cub-sfGFP (8) 

empty 

empty 

empty 

empty 

Confirmed 

CD22 

For succellular localization assay of sIL-6R-Nub 

empty 

empty 

empty 

sIL-6R-Nub-sfGFP (10) 

empty 

Confirmed 

CD23 

For succellular localization assay of sIL-1R2-Cub 

sIL-1R2-Cub-sfGFP (12) 

empty 

empty 

empty 

empty 

Confirmed 

CD24 

For succellular localization assay of sIL-1RAcP-Nub 

empty 

empty 

empty 

sIL-1RAcP-Nub-sfGFP (14) 

empty 

no 

CD25 

For succellular localization assay of sIL-10R1-Cub 

sIL-10R1-Cub-sfGFP (16) 

empty 

empty 

empty 

empty 

Confirmed 

CD26 

For succellular localization assay of sIL-10R2-Nub 

empty 

empty 

empty 

sIL-10R2-Nub-sfGFP (18) 

empty 

Confirmed 

CD27 

For succellular localization assay of sgp130(D1-D6)-cTEV 

sgp130(D1-D6)-cTEV-sfGFP (22) 

empty 

empty 

empty 

empty 

Confirmed 

CD28 

For succellular localization assay of sgp130(D1-D6)-nTEV 

empty 

empty 

empty 

sgp130(D1-D6)-nTEV-sfGFP (23) 

empty 

Confirmed 

CD29 

For succellular localization assay of sgp130(D1-D3)-cTEV 

sgp130(D1-D3)-cTEV-sfGFP (26) 

empty 

empty 

empty 

empty 

no 

CD30 

For succellular localizationassay of sIL-6R-nTEV 

empty 

empty 

empty 

sIL-6R-nTEV-sfGFP (27) 

empty 

Confirmed 

CD31 

For succellular localization assay of sIL-1R2-cTEV 

sIL-1R2-cTEV-sfGFP (30) 

empty 

empty 

empty 

empty 

no 

CD32 

For succellular localization assay of sIL-1RAcP-nTEV 

empty 

empty 

empty 

sIL-1RAcP-nTEV-sfGFP (31) 

empty 

Confirmed 

CD33 

For succellular localization assay of sIL-10R1-cTEV 

sIL-10R1-cTEV-sfGFP (34) 

empty 

empty 

empty 

empty 

no 

CD34 

For succellular localization assay of sIL-10R2-nTEV 

empty 

empty 

empty 

sIL-10R2-nTEV-sfGFP (35) 

empty 

Confirmed 

CD35 

For succellular localization assay of sIL-6R 

empty 

empty 

empty 

sIL-6R-sfGFP (6) 

empty 

no 

CD36 

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 

CD12.01 

A2A biosensor with GPA1-mutation 1 

A2A (R199A) 

GPA1-mutation 1 

LexA-Ste12 

empty 

LexAop-nanoluc 

no 

CD12.02 

A2A biosensor with GPA1-mutation 2 

A2A (R199A) 

GPA1-mutation 2 

LexA-Ste12 

empty 

LexAop-nanoluc 

no 

CD12.03 

A2A biosensor with GPA1-mutation 3 

A2A (R199A) 

GPA1-mutation 3 

LexA-Ste12 

empty 

LexAop-nanoluc 

no 

CD12.04 

A2A biosensor with GPA1-mutation 4 

A2A (R199A) 

GPA1-mutation 4 

LexA-Ste12 

empty 

LexAop-nanoluc 

no 

CD12.05 

A2A biosensor with GPA1-mutation 5 

A2A (R199A) 

GPA1-mutation 5 

LexA-Ste12 

empty 

LexAop-nanoluc 

no 

CD13 

A2A biosensor 

A2A (R199A) 

GPA1 

LexA-Ste12 

empty 

LexAop-nanoluc 

no 

CD14.01 

A2A biosensor with GPA1-mutation 1 and inducible TEV protease 

A2A (R199A) 

GPA1-mutation1 

LexA-Ste12 

scGal1-TEV 

LexAop-nanoluc 

Confirmed 

CD14.02 

A2A biosensor with GPA1-mutation 2 and inducible TEV protease 

A2A (R199A) 

GPA1-mutation2 

LexA-Ste12 

scGal1-TEV 

LexAop-nanoluc 

Confirmed 

CD14.03 

A2A biosensor with GPA1-mutation 3 and inducible TEV protease 

A2A (R199A) 

GPA1-mutation3 

LexA-Ste12 

scGal1-TEV 

LexAop-nanoluc 

Confirmed 

CD14.04 

A2A biosensor with GPA1-mutation 4 and inducible TEV protease 

A2A (R199A) 

GPA1-mutation4 

LexA-Ste12 

scGal1-TEV 

LexAop-nanoluc 

no 

CD14.05 

A2A biosensor with GPA1-mutation 5 and inducible TEV protease 

A2A (R199A) 

GPA1-mutation5 

LexA-Ste12 

scGal1-TEV 

LexAop-nanoluc 

no 

CD15 

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 

CD10 

TMD-LexA-VP16 cleaved by inducible TEV 

empty 

TMD-LexA-VP16 (44) 

empty 

scGal1-TEV 

LexAop-nanoluc 

Confirmed 

CD37 

Leucinezippers (K3 and E3) with partially optimised cTEV as in Bba_K1159102  

(36.1) 

TMD-LexA-VP16 (44) 

empty 

(37.1) 

LexAop-nanoluc 

no 

CD38 

Leucinezippers (K3 and E3) with most optiimsed cTEV 

(38.1) 

TMD-LexA-VP16 (44) 

empty 

(37.1) 

LexAop-nanoluc 

no 

CD39 

Leucinezippers (K3 and E3) with Wt cTEV as in Bba_K1965020  

(39.1) 

TMD-LexA-VP16 (44) 

empty 

(37.1) 

LexAop-nanoluc 

no 

CD40 

Leucinezippers (ZR and ZE) with partially optimised cTEV as in Bba_K1159102  

(36.2) 

TMD-LexA-VP16 (44) 

empty 

(37.2) 

LexAop-nanoluc 

no 

CD41 

Leucinezippers (ZR and ZE) with optimised cTEV   

(38.2) 

TMD-LexA-VP16 (44) 

empty 

(37.2) 

LexAop-nanoluc 

no 

CD42 

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 

CD11 

control for reporter leakage   

empty 

empty 

empty 

empty 

LexAop-nanoluc 

no 

Table 2: Yeast strains


Minimal biosensor design

All our biosensor designs are based on the association of extracellular domains of receptor proteins in the presence of interleukins at the plasma membrane of S. cerevisiae.

In the minimal design, the extracellular Interleukin-receptors are fused with either a C- or N-terminal part of a split-ubiquitin protein. Upon receptor association in the presence of IL-molecules, the two halves reconstitute resulting in full-length ubiquitin that can be recognized by a deubiquitinase leading to cleavage of the protein and release of a LexA-VP16 synthetic transcription factor. This would further result in the expression of the luciferase reporter gene (nanoluc).
Figure 8: Minimal biosensor design; Split ubiquitin complementation leading to release of C-ub bound LexA-VP16 to initiate gene expression.

Induction assays

In order to test whether our first design of the biosensor works, induction assays with dilution series of the corresponding interleukins were carried out.

Biosensors for IL-10 and IL-6, corresponding to assembly 1 and 2 in table 2, were tested. The two strains were grown to an OD600=0,5 and incubated at 30°C for a set of different incubation times with different concentrations of the respective biomarker. Next, luciferase assays were performed with the strains (figures 9 and 10).
Figure 9: Luciferase assay of the minimal interleukin-6 biosensor. The amount of luminescence observed after incubation with a dilution series of interleukin-6 for 1, 3, 14 and 22 hours.
Figure 10: Luciferase assay of the minimal interleukin-10 biosensor. The amount of luminescence observed after incubation with a dilution series of interleukin-10 for 1, 3, 14, 22 and 44 hours.
No correlation between interleukin concentration and luminescence was observed for either strain at any incubation time. Thus, the biosensors did not function at the given interleukin concentrations in the dilution series, under the conditions of the experiment.
Interestingly, we noticed that the level of luminescence obtained for the IL-6 biosensor is 3-10 times higher than for the IL-10 biosensor. This suggests that the IL-6 biosensor might be constitutively signalling. One possible explanation is that there was affinity between the two receptor proteins even when the interleukin was not present. This would further infer that the two receptor proteins were localized to the same subcellular compartments. Alternatively, the receptor protein with the transcription factor had been partially degraded which would allow the transcription factor to activate expression of the reporter.

For both biosensors, the amount of luminescence varied over time with the highest intensity recorded after 14 hours of incubation and lower intensity at shorter and longer incubation times. This might reflect that the cells were in different growth phases at the different times of the luciferase assays.

Subcellular localization of receptors

In order for our biosensors to work, the receptor proteins need to be localized to the plasma membrane of S. cerevisiae. To investigate whether the subcellular localization of the proteins could be the explanation for the dysfunction of our biosensors, confocal fluorescence microscopy was carried out.

Yeast strains that constitutively express super-folding green fluorescent protein (sfGFP) linked to the C-terminal of the receptor proteins were constructed (see table with strains for localization assays) to track the localization of the proteins. After confirming the strains for chromosomal integration of the receptor-GFP fusion constructs, the strains were grown in liquid media and observed with confocal fluorescence microscopy.
IL-6 receptor proteins
Flourescence Brightfield
sIL-6R-Nub-sfGFP
sgp130(D1-D3)-Cub
Figure 11a: Subcellular localization of sgp130(D1-D3)-Cub Confocal laser scanning microscopy suggests possible localization of sgp130(D1-D3)-Cub in the endoplasmic reticulum (a), with some protein also localizing to the plasma membrane (b). Inclusion bodies/vacuoles (c) are visible on the brightfield image.





Figure 11b: Subcellular localization of sIL-6R-Nub The majority of sIL-6R-Nub-sfGFP localizes to inclusion bodies (a) near the plasma membrane of Saccharomyces cerevisiae cells. Little to none of the protein can be found localized to other subcellular compartments, with only faint localization uniformly in the cytosol (c) and plasma membrane (b).
Confocal laser scanning microscopy suggests that both sgp130-Cub and IL-6R-Nub exhibit low degrees of localization to the plasma membrane, yet none of them localize there perfectly. Both localize in greater abundance in the endoplasmic reticulum. IL-6R-Nub seemed to have a higher preference for localization in inclusion bodies or cytosol. This apparent overlap in localization of sgp13-Cub and IL-6R-Nub could confirm the hypothesis inspired by results from luciferase assays, i.e., that both proteins are localized in the same compartment, which would be a necessary prerequisite for them to bind even in absence of IL-6.

However, findings from the confocal fluorescence microscopy cannot lead to rejection of the other hypothesis – that the system is always signalling due to the partial degradation of one of the proteins, thus, allowing the transcription factor to reach the nucleus at all times. Results from the confocal fluorescence microscopy indicate that the problems with the dysfunctional biosensor might be caused by unsatisfactory localization to the plasma membrane.

IL-10 receptor proteins
Flourescence Brightfield
sIL-10R1-Cub-sfGFP
sIL-10R2-Nub-sfGFP
Figure 12a: subcellular localization of sIL-10R1-Cub. sIL-10R1-Cub-sfGFP is shown to primarily localize to the plasma membrane (a) of S. cerevisiae cells and faintly to moderately in the endoplasmic reticulum (b). Small inclusion bodies (c) can be observed near the plasma membrane.





Figure 12b subcellular localization of sIL-10R2-Nub-sfGFP. sIL-10R2-Nub-sfGFP shows localization within the entirety (a) of the S. cerevisiae cell and the plasma membrane (b) without distinction of which subcellular compartments that the protein resides in.

According to confocal microscopy, sIL-10R1-Cub seemed to localize in the endoplasmic reticulum, plasma membrane and inclusion bodies. However, localization of sIL-10R2-Nub in the cell was uniform, or with a preference for the cytosolic and nuclear compartments. According to the luciferase assay, the biosensor was not functioning. Localization of sIL-10R2-Nub is a highly likely reason for dysfunction, as both sIL-10R1-Cub and sIL-10R2-Nub must be present at the plasma membrane in order to associate in the presence of IL-10 and trigger intracellular signalling.

As a conclusion, confocal fluorescence microscopy, together with luciferase assays, show that improving protein localization of the IL-10 receptor could be a plausible way to improve the IL-10 biosensor. However, causes of spontaneous signalling of the IL-6 biosensor need to be elucidated to guide further modifications of the biosensor.

Intermediate biosensor design

In this design, further amplification of the biosensor signal is achieved by adding an enzymatic step to the signaling pathway. Whereas in the minimal design, one pair of activated receptors can only release one transcription factor, in this design one activated receptor pair can lead to the release of multiple membrane bound transcription factors. Here, the extracellular IL-receptors were fused with the Wsc1 transmembrane domain and either the C- or N-terminal part of a split-TEV protease. Upon receptor association in the presence of IL molecules, the two halves reconstitute to a functioning TEV protease. This protease recognizes the membrane bound transcription factor LexA-VP16 and releases it from the membrane.
Figure 13: Intermediate biosensor design. Split-TEV protease complementation leading to cleavage of a TEV-recognition sequence near LexA-VP16, thereby, releasing LexA-VP16 to initiate gene expression.

Localization of the receptors

In order for the receptor proteins of the intermediate and the advanced design to work, they needed to be localized to the plasma membrane of S. cerevisiae. Subcellular localization seemed to be part of the cause that the biosensors of the minimal design did not work. Therefore, localization assays of the receptor proteins for the intermediate and advanced design were carried out to gauge the chance of success for these designs.

Strains constitutively expressing superfolder green fluorescent protein linked to the C-terminal of the receptor proteins were created (table 2), grown and observed with confocal laser scanning microscopy.
Receptor proteins
Flourescence Brightfield
sIL-6R-nTEV-sfGFP
sIL-1RAcP-nTEV-sfGFP
sIL-10R2-nTEV-sfGFP
Figure 14a subcellular localization of sIL-6R-nTEV. sIL-6R-nTEV-sfGFP can be observed to likely localize primarily to the plasma membrane (a), both in small and medium-sized inclusion bodies (b). Some protein localizes in the endoplasmic reticulum (c).











Figure 14b subcellular localization of sIL-1RAcP-nTEV sIL-1RAcP-nTEV-sfGFP seemingly localizes uniformly throughout the cytosol (a) of the cell in what looks like inclusion bodies, seemingly without localizing, or localizing very little, to the plasma membrane (b).










Figure 14c subcellular localization of sIL-10R2-nTEV sIL-10R2-nTEV-sfGFP localizes primarily to inclusion bodies (a) 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 is also localized within the cytosol (b). While unclear, it may seem that some of the protein localizes within the endoplasmic reticulum (c).

sIL-6R-nTEV localized preferentially to the plasma membrane, unlike sIL-1RAcP-nTEV which had a preference for the cytosolic and nuclear compartments. sIL-10R2-nTEV localized to the membrane in the form of inclusion bodies, but also localized within the cytosol.

sIL-6R-nTEV seems to localize as desired and could be possibly used as part of a IL-6 biosensor. In contrast, improvement in the localization of sIL-10R2-nTEV may be needed before it can perform its role in a IL-10 biosensor. Likewise, localization of sIL-1RAcP-nTEV needs to be significantly improved before it can function in a biosensor for IL-1.

Cleavage of membrane bound transcription factor

The results from the subcellular localization assays and the induction assays of the minimal design indicated that the receptor proteins of the intermediate and advanced design would have a poor chance of success without further optimization of subcellular localization and the design of the receptor proteins in general. Due to limited time, we chose to focus on characterizing the accessory proteins of the intermediate and advanced design, i.e. the membrane bound transcription factor in the intermediate design and the modified GPA1 proteins in the advanced design.

In the intermediate biosensor design, a split TEV protease releases transcription factors from the membrane upon receptor association and TEV reconstitution. For this, we constructed a transmembrane protein - 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 constitutively expressed accessory protein, an inducible TEV protease and a nanoLuc luciferase under the control of a synthetic LexAop promoter into S. cerevisiae as described above (see assembly 10 in table 2). The strain was grown to an OD600 of 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. Then, a luciferase assay was carried out with the culture.
Figure 15: Luminescence assay based on cleavage of membrane bound transcription factor. Cells were expressed without (glucose media) and with (galactose+raffinose media) induction of the TEV protease. A 1300-fold increase in luminescence was seen upon induction of the TEV protease. Data shown is the average of three biological replicates.

Advanced biosensor design

In the advanced design, amplification of the biosensor signal is achieved by hijacking the yeast pheromone pathway through a modified Gα subunit. The IL-receptors were fused with halves of a split-TEV protease that can cleave a modified GPA1 subunit. Upon association of the receptors, the two halves will reconstitute and cleave Gα, releasing the beta-gamma subunit that transduces the signal.
Figure 16: Advanced biosensor design. Split-TEV protease complementation leading to cleavage of a TEV protease cut site in GPA1, thereby, initiating gene expression through the yeast pheromone pathway.

Testing functionality of modified GPA1

For the third design to work, the modified GPA1 must be cleaved by the TEV protease, dissociate from the Gβγ complex and induce signalling through the pheromone pathway. This experiment was designed to test whether expression of a TEV protease, induced by galactose media, could activate signalling through the pheromone pathway. The experiment was further designed to investigate whether the native functions of GPA1 were conserved in the modified GPA1 proteins either with or without induction of a TEV protease. For more information about the different versions of GPA1, visit the modelingand engineering successpages.

The different GPA1 mutants were tested in the context of an adenosine biosensor employing the human adenosine GPCR receptor A2A(R199A) (unpublished) in S. cerevisiae, previously developed in the Kampranis lab. Compared to the original biosensor, the strains in this experiment had an inducible TEV protease and the wt GPA1 was exchanged with the modified GPA1 proteins (see table 2, strains 14.1-14.3 and 15). The strains were grown to an OD600 of 0.4 and incubated in either glucose or galactose + raffinose media at 30°C for 17 hours. Next, the amount of signalling was measured with a luciferase assay.
Figure 17: Characterization of modified GPA1 proteins in adenosine biosensor. The biosensors were grown in either glucose media or galactose + raffinose media which repressed or activated expression of a TEV protease. The biosensors were further grown with a dilution series of adenosine.

For all versions of GPA1, and at all concentrations of adenosine, the amount of luminescence was higher when cells were incubated in galactose + raffinose media compared to glucose media. This was also the case for the biosensor without any TEV cleavage site introduced to the GPA1 sequence. This infers that the rise in luminescence when the media was changed might be caused by other mechanisms than the cleavage of GPA1. Thus, the experiments neither confirmed nor rejected that the TEV protease was able to activate signalling through the cleavage of the GPA1 mutants.

Biosensors with version one and three of the GPA1 subunit showed very high amounts of luminescence compared to the biosensor with wt GPA1 even in glucose media. This might suggest that these versions of GPA1 are always causing signalling through the pheromone pathway. Mutation 2 showed very low amounts of luminescence compared to the biosensor with wt GPA1 even in galactose media. This might suggest that the Gα strongly inhibits all signalling, that the pheromone pathway has been shut down, or that the yeast has mutated so that the reporter expression is uncoupled from the pheromone pathway.

For the biosensor with wt GPA1, a correlation between adenosine concentration and luminescence was observed at a level that reproduced earlier results from the Kampranis lab when glucose was used as a carbon source. A similar tendency was not observed for any of the mutant GPA1 strains neither with glucose nor galactose + raffinose media. This infers that all the mutations cause a loss-of-function in the sense that the GPA1 is not able to transduce the signal from the A2A GPCR.

The comparative experiment of effects of glucose media and galactose + raffinose media on mutant GPA1 strains and the wt GPA1 strain yielded mixed results. It was not possible to confirm nor reject whether TEV protease can trigger signalling via cleavage of mutant GPA1. However, the experiment did show that mutant GPA1 strains lost the ability to transduce signal from the A2A GPCR, while it was maintained in the wildtype GPA1.

Conclusion/discussion

To sum up our achievements in the laboratory, we managed to:
  • ✧ Test the functionality of our minimal design biosensor
  • ✧ Investigate the subcellular localization of 7 of our receptor proteins
  • ✧ Verify the functionality of the accessory protein, the membrane bound synthetic transcription factor, in the intermediate design.
  • ✧ Test various functions of the modified GPA1 proteins for the advanced design, eg. the ability to signal after being cleaved by a TEV protease and the ability to transduce the signal from a GPCR.
  • ✧ Clone 54 constructs
  • ✧ Create 21 new yeast strains

Our induction assays showed that the IL-6 and IL-10 biosensors of our minimal design were not able to sense different concentrations of the respective biomarkers. For the interleukin-6 biosensor, the system seemed to be constantly signalling. This might indicate that the dysfunctionality could, to some extent, be explained by an affinity between the receptor proteins in the absence of IL-6. To understand why our biosensors were dysfunctional, the subcellular localization of our modified receptors and accessory proteins were investigated using confocal fluorescence microscopy. In order for our receptor proteins to work, they had to be localized at the plasma membrane of S. cerevisiae. While none of the modified proteins solely localized to the plasma membrane, they were all expressed to some extent, some more than others. For the minimal design, sgp130(D1-D3)-Cub-sfGFP only localized faintly to the plasma membrane, while the soluble sIL-6R-Nub-sfGFP almost solely was found in inclusion bodies. This might explain the dysfunctionality of the minimal IL-6 biosensor.

sIL-10R1-Cub-sfGFP localized primarily to the plasma membrane and endoplasmic reticulum but sIL-10R2-Nub-sfGFP localized unspecifically to the entire cell, mainly in the cytosolic compartment. This might, therefore, also explain the dysfunctionality of the minimal IL-10 biosensor.

The subcellular localization assays of sIL-6R-nTEV-sfGFP, sIL-1RAcP-nTEV-sfGFP, and sIL-10R2-nTEV-sfGFP all showed cytosolic localization and the presence of inclusion bodies, with only sIL-6R-nTEV-sfGFP localizing primarily to the plasma membrane in cells lacking inclusion bodies. However, this was seen for cells containing vacuoles, which may not be representative for the entire population of cells.

For the intermediate design, it was evident that the transcription factor in the form of LexA-VP16 containing a transmembrane domain and a TEV-recognition site in a flexible linker, was able to be cleaved by a TEV protease. Upon induction of TEV protease expression, and carrying out a luciferase assay, luminescence was increased over 1000-fold. This indicated that the protein functioned as intended. In hindsight, the experiment could have been designed to include a control strain without the inducible TEV protease as well as a strain without the membrane bound transcription factor. A western blot of a tagged version of the protein after incubation in either media could further verify that the protein is cleaved by the TEV protease. The next step in characterizing the membrane bound transcription factor would be to investigate whether it can be cleaved by a membrane bound split TEV protease.

For the advanced design, the functionality of the modified GPA1 proteins was compared to the functionality of the wt GPA1 in an adenosine biosensor. To test how cleavage of the modified GPA1 proteins affected signalling through the pheromone pathway, expression of a TEV protease was controlled by changing media from glucose to galactose. The signalling increased in all the modified GPA1 proteins as expected. Surprisingly, the biosensor with wt GPA1 also showed increased signalling upon change of the media. It is, therefore, inconclusive whether the GPA1 proteins functioned as intended. To shed further light on the functionality of the GPA1 proteins, the experiment would have to be designed differently. This could include making a control strain without the TEV protease or utilizing another method for inducing expression of the TEV protease where the carbon source is kept constant. One possible explanation for the elevated level of luminescence with galactose media is that the pPGK1 promoter, which regulates the expression of the GPA1 protein, might be less active when cells are grown in galactose media. Since GPA1 is inhibiting signalling through the pheromone pathway when bound to the Ste4-Ste18(Gβγ)-complex, less GPA1 present would lead to more signalling through the pathway.

Finally, the ability of the modified GPA1 proteins to transduce a signal from a GPCR was investigated. While the results showed that the adenosine biosensor with wt GPA1 responded to the adenosine concentrations as expected, this response was not observed for any of the adenosine biosensors with modified GPA1 proteins. Thus, the modifications introduced a loss-of-function in respect to signal transduction in GPCR-based biosensors. The investigations as to whether the modifications introduced a gain-of-function in regards to signalling through cleavage by the TEV protease were inconclusive.



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