Identifying Transmembrane Interactions in Receptor Protein Tyrosine Phosphatase Homodimerization and Heterodimerization

Abstract

Receptor protein tyrosine phosphatases (RPTPs) are one of the key regulators of receptor tyrosine kinases (RTKs) and therefore play a critical role in modulating signal transduction. While the structure-function relationship of RTKs has been widely studied, the mechanisms modulating the activity of RPTPs still need to be fully understood. On the other hand, homodimerization has been shown to antagonize RPTP catalytic activity and appears to be a general feature of the entire family. Conversely, their documented ability to physically interact with RTKs is integral to their negative regulation of RTKs. But there is yet to be a proposed common model. However, specific transmembrane domain (TM) interactions and residues have been shown to be essential in regulating RPTP homo-dimerization, interactions with RTK substrates, and activity. Therefore, elucidating the contribution of the TM domains in RPTPs regulation can provide significant insights into how these receptors function, interact, and eventually be modulated. This chapter describes the dominant-negative transcriptional activator-based (DN-AraTM) assay to identify specific TM interactions essential to homodimerization and hetero association with other membrane receptors, such as RTKs.

1. Introduction

Twenty-two human protein tyrosine phosphatases (PTPs) are classified as receptor protein tyrosine phosphatases (RPTPs). They all share the same architecture: an extracellular region, a single-pass transmembrane segment, and one or two cytosolic and highly-conserved PTP domains [1]. By being some of the most important regulators of receptor tyrosine kinases (RTKs), RPTPs play critical signaling regulatory roles in development, homeostatic control, health, and disease progression. They can be tumor-suppressors of many cancers, including colon, lung, breast, and thyroid. Still, they can also lead to cancer progression when deregulated through reduced expression or loss-of-function mutations [2–6]. Therefore, RPTPs are crucial for controlling cell growth and have long been viewed as potential therapeutic targets. Yet, the detailed structural basis underpinning the regulation of their catalytic activity remains unclear.

However, the activity of many RPTPs has been reported to be suppressed by homodimerization, which may prevent RPTP access to their RTK substrates. The transmembrane (TM) domains for RPTPs are proposed to mediate homodimerization, although there is no clear structure-based model for this mechanism [7–10]. Understanding the influence of RPTP TM domains in oligomerization and activity could advance our basic biological understanding of how RPTPs regulate cell signaling pathways in cells and develop new therapeutic approaches to promote RPTP activity against their oncogenic RTK substrates.

We previously reported that specific TM interactions and residues regulate the homodimerization of a member of the RPTP family (protein tyrosine phosphatase receptor J or PTPRJ) [11]. To do so, we used the dominant-negative AraC-based transcriptional reporter assay (DN-AraTM), which reports on the propensity of TM domains to self-associate and heterodimerize in cell membranes. Disrupting these interactions, through point mutations in the TM domains of the full-length receptor expressed in mammalian cells, disrupted PTPRJ homodimerization, reduced access to one of its substrates EGFR [12–14], led to reduced EGFR phosphorylation, and antagonized EGFR-driven cell phenotypes [11]. Similar results have been obtained in acute myeloid leukemia cell models expressing the PTPRJ mutant and the oncogenic FLT3 mutant (with internal tandem duplications) [15].

In this chapter, we describe the use of the DN-AraTM assay to readily identify TM-TM interactions mediating RPTP homodimerization and hetero-association with other single-pass membrane receptors, like RTKs. Briefly, the assay relies on protein chimeras containing the receptor TM domains of interest fused to either the E. coli transcription factor AraC (active at the araBAD promoter as a homodimer) or to its inactive R210A mutant (namely AraC*) (Fig. 1a) [16, 17]. Both chimeras include an N-terminal maltose-binding protein (MBP) fusion to promote unidirectional insertion in the inner membrane of AraC-deficient E. coli (with MBP residing in the periplasm). The AraC and AraC* also include an HA-tag and myc-tag, respectively, to determine their expression levels. TM-mediated homodimerization of AraC induces the transcription of the green fluorescent protein (GFP) reporter gene under the control of the araBAD promoter, enabling quantification of fluorescence measurements on whole cells directly from culture [16–19]. On the other hand, preferential TM-mediated AraC-AraC* heterodimerization reduces the level of GFP transcription. Therefore, the level of GFP fluorescence intensity, when normalized for cell density and expression level, is a measure of receptor domain dimerization (Fig. 1b). Depending on which TM domain is in fusion with AraC or AraC*, the DN-AraTM assay enables simultaneous measurement of homodimerization and heterodimerization. Notably, the effect of point mutations on both processes can be evaluated.

Figure 1.

(a) Overview of the DN-AraTM assay. Chimera containing N-terminal MBP fused to either an in-frame receptor A fragment (Domain A; dark blue) and C-terminal AraC or an in-frame receptor B fragment (Domain B; light blue) and C-terminal disabled AraC unable to activate transcription at the araBAD promoter (AraC*) are expressed by the regulator plasmids (pAraTMwt, ampicillin-resistant; pAraTMDN, kanamycin-resistant). Once expressed, MBP directs the integration of chimera in the inner membrane of E. coli. Homodimerization of Domain A (AraC-AraC) brings the AraC transcription factors in close proximity and activates the araBAD promoter to produce GFP. If Domain A has a higher affinity to heterodimerize with Domain B versus homodimerize with Domain A, a reduction in GFP will be observed (AraC-AraC*) due to the inability of heterodimers containing receptor B-fused AraC* to activate transcription at the araBAD promoter. This figure was created with Biorender.com). (b) Representative example of results after correction for bacterial growth rate and chimera expression levels if needed (bars are color-coded as in (a).

2. Materials

2.1. Subcloning the Transmembrane domain of interest into AraTM plasmids

1. AraTM Plasmids: pAraTMwt (AraC plasmid; Addgene # 47514) and pAraTMDN (AraC* plasmid; Addgene # 47515)

2. Oligonucleotide primers to amplify and subclone the DNA sequence coding TM domains of interest into the AraTM plasmids (AraC/AraC*). The final insert should include SacI and KpnI restriction sites at the 5’ and 3’, respectively (see Notes 1 and 2).

3. DNA Polymerase.

4. SacI and KpnI restriction enzymes.

5. T4 DNA ligase with appropriate buffer.

6. E. coli DH5α competent cells for transformation (or equivalent).

7. Sequencing primer to confirm subcloning (see Note 3).

8. LB medium with appropriate selective antibiotic (100 µg/mL Ampicillin or 50 µg/mL Kanamycin).

9. LB agar plate with appropriate selective antibiotic (100 µg/mL Ampicillin or 50 µg/mL Kanamycin) (see Note 4).

10. Plasmid DNA purification kit.

11. Plasmid DNA gel extraction kit.

12. Thermocycler.

13. 0.8% agarose gel.

14. Agarose gel electrophoresis apparatus.

15. UV light transilluminator.

2.2. Co-Transformation of AraC, AraC*, and Reporter Plasmids into E. coli

1. AraC-deficient E. coli strain SB1676 (The E. coli Genetic Stock Center at Yale University).

2. Empty vectors: pAraTMwt(empty) and pAraTMDN(empty) (not available on Addgene)

3. pAraGFPCDF (GFP reporter plasmid; Addgene # 47516).

4. LB medium and agar plates with appropriate selective antibiotics.

2.3. DN-AraTM Assay and Analysis

1. 24-well deep well plate (10 mL) with U-bottom for bacterial growth and autoclave safe (see Note 5).

2. Adhesive and porous film for culture plates (e.g., VWR cat#60941–086)

3. LB medium (100 µg/mL Amp, 50 µg/mL Kan, Spec 100 µg/mL).

4. LB agar plate (100 µg/mL Amp, 50 µg/mL Kan, Spec 100 µg/mL).

5. ZY media: 1% (w/v) tryptone, 0.5% (w/v) yeast extract.

6. 5052 (50x): 25% glycerol, 2.5% (w/v) glucose, 10% (w/v) ɑ-lactose.

7. P(NPS) (20x): 1M sodium phosphate dibasic Na₂HPO₄, ~ pH 6.75.

8. Autoinduction media (100 mL): 92.8 mL of ZY media, 1× 5052, 1x P(NPS), and 2 mM MgSO₄.

9. Assay plate, 96-well, black with clear and flat bottom, polystyrene (e.g., Corning cat# 3631)

10. Plate shaker with temperature adjustment.

11. Plate reader with filters for measuring absorbance at 600 nm and GFP fluorescence emission (i.e., 488 nm and 510 nm for excitation and emission, respectively).

2.4. Confirming Protein Expression Level by Immunoblotting

1. 8M urea.

2. 4x SDS loading dye: 14 mM Tris-HCl, pH 6.8, 17.2 mM β-mercaptoethanol, 5% glycerol, 8% (w/v) SDS, 0.015% (w/v) bromophenol blue (see Note 6).

3. Heat Block.

4. PAGE apparatus.

5. 8% SDS-Polyacrylamide gel (homemade or from a commercial source).

6. SDS-PAGE running buffer (1L): 2.5 mM Tris base, pH 8.6, 19.2 mM Glycine, 0.1% (w/v) SDS.

7. Transfer buffer (2L): 5 mM Tris base, pH 8.6, 38.4 mM Glycine, 20% methanol.

8. Nitrocellulose blotting membrane (e.g., Cytiva cat# 1060003).

9. Transfer apparatus.

10. 5% (w/v) bovine serum albumin solution (BSA)

11. Rocking Platform.

12. Tris-buffered saline (TBS; 1x): 1 mM Tris base, pH 7.6, 15 mM NaCl.

13. TBS containing 0.1% Tween-20 (TBS-T).

14. Blocking solution: 5% milk in TBS-T or commercially available blocking solution.

15. Antibodies: Antibodies: HRP-conjugated anti-MPB antibody (1:10000; New England Biolabs cat#E8038, RRID:AB_1559738), HRP-conjugated anti-Myc (1:5000; Cell Signaling Technology cat# 2040, RRID:AB_2148465), and anti-HA (1:5000; Thermo Fisher Scientific cat# 26183, RRID:AB_10978021) followed by secondary anti-mouse HRP-conjugated (1:5000; Cell Signaling Technology cat# 7076, RRID:AB_330924).

16. Chemiluminescent horseradish peroxidase (HRP) substrate (e.g., Bio-Rad cat# 170–5061).

17. Gel imaging system capable of detecting chemiluminescence.

18. Image analysis software (e.g., ImageJ).

2.5. Confirm Chimera Insertion Topology by Maltose Complementation Assay

1. MBP-deficient MM39 E. coli strain [20, 21].

2. 5x M9 Minimal Salts: 210 mM Na₂HPO₄, 110 mM KH₂PO₄, 42.8 mM NaCl, 93 mM NH₄Cl.

3. Agar.

4. 1M MgSO₄ (filter sterilized).

5. 1M CaCl₂ (filter sterilized).

6. 50% Glucose (filter sterilized).

7. 1000x Ampicillin solution: 100 mg/mL (filter sterilized).

8. 1000x Kanamycin solution: 50 mg/mL (filter sterilized).

2.6. Confirming Chimera Insertion Topology by Spheroplast Protection Assay

1. Periplasting Buffer: 200 mM Tris-HCl, pH 7.5, 20% sucrose, 1mM EDTA, and 30 U/µL Ready-Lyse Lysozyme Solution (e.g., VWR, Catalog #76081–780)

2. Lysis Buffer: 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM EDTA, and 0.1% deoxycholate.

3. DNA/RNA non-specific endonuclease (e.g., OmniCleave Endonuclease. 50,000 U)

4. Ultra-pure water

5. 1.0 M MgCl₂ solution

6. 500 mM EDTA solution.

7. Proteinase K (e.g., New England Biolabs cat# 8107)

8. Nonidet P-40 (NP-40) (e.g., Thermo Scientific cat# 28324)

3. Methods

3.1. Subcloning the Transmembrane domain of interest into AraTM plasmids

1. Amplifies the transmembrane domains of interest with designed primers following standard PCR protocol (see Note 7).

2. Digest both transmembrane domains of interest alongside the AraC and AraC* plasmid backbones with SacI and KpnI restriction enzymes.

3. Run the digested product on an 0.8% agarose gel by electrophoresis (see Note 8).

4. Gel purify the appropriate bands using a DNA gel extraction kit and following the manufacturer’s protocol.

5. Ligate the purified insert and vectors using T4 DNA ligase, following standard molecular biology techniques (see Note 9).

6. Transform in E. coli DH5α competent cells and grow using appropriate antibiotics (see Note 4).

7. Confirm via sequencing using the AraC-seq primer.

3.2. Co-Transformation of AraC and AraC* Plasmids into E. coli

Specific pairs of co-transformations are required depending on whether homo- or hetero-association is being assessed (Tables 1 and 2) (see Note 10).

Table 1.

Setup for co-transforming SB1676 E. coli with the GFP reporter, AraC and AraC* plasmids.

	Homodimerization Domain A			Homodimerization Domain B			Heterodimerization A+B
	1	2	3	4	5	6	7	8
AraC	domain A	domain A	empty vector	domain B	domain B	empty vector	domain A	domain B
AraC*	empty vector	domain A	domain A	empty vector	domain B	domain B	domain B	domain A
GFP reporter	+	+	+	+	+	+	+	+

Table 2.

Example of co-transformation setup to study the effect of point mutation on TM self-association.

	Homodimerization Domain A
	1	2	3
AraC	wild-type	TM mutant 1	TM mutant 2
GFP reporter	+	+	+

1. Co-transform SB1676 cells with 200–500 ng of pAraGFPCDF, AraC and AraC* plasmids according to Tables 1 and 2, following standard transformation protocol (see Note 11).

2. Grow on an LB selective plate (100 µg/mL Amp, 50 µg/mL Kan, Spec 100 µg/mL) (see Note 12).

3.3. DN-AraTM Assay and Analysis

The overall protocol is represented in Figure 2.

Figure 2.

Overview of the protocol’s key steps (created on Biorender.com).

1. Add 2 mL of LB selective medium to the appropriate number of wells in a 24-well deep well plate (see Note 13).

2. Pick individual colonies with a pipette tip.

3. Place each tip directly into individual wells.

4. Cover the plate with adhesive film.

5. Incubate at 30°C for 8 hours with shaking (50 rpm on a plate shaker).

6. In a new 24-well deep well plate, add 2 mL of auto-induction media to the correct number of wells.

7. Take 20 µL of culture from each well of the 24-well growth plate, and transfer to individual wells on the 24-well plate.

8. Cover the 24-well deep well plate and incubate overnight at 30°C with continuous shaking at 50 rpm (see Note 14).

9. In a 96-well assay plate (black with clear flat bottom), perform two-fold serial dilutions (100 µL total volume) for each culture using ZY media (undiluted, 1:2, 1:4, and 1:8). (see Notes 15 and 16).

10. Measure the absorbance at 600 nm and GFP fluorescence emission at 510 nm using a plate reader.

11. For each sample, subtract the absorbance and fluorescence values obtained with media alone.

12. Graph fluorescence emission to absorbance for each sample and dilution (Figure 3a).

13. Determine the slope for each sample.

14. Represent slopes as a bar graph (Figure 3b).

Figure 3.

(a) fluorescence intensity at 510 nm of different pair combinations from serial dilutions of bacterial cultures plotted against the corresponding cell density (absorbance at 600 nm). Results are shown as mean ± S.E. (n=9). Solid lines represent the best-fit line through the experimental data, and the shaded areas represent the upper and lower bound of the 95% confidence interval for each fit. (b) GFP signal is normalized to the signal of the sample (1), and the results are shown as mean ± S.E. (n=9). Results were normalized, taking into account the slight differences in expression level measured by immunoblotting. The level of significance (one-way ANOVA with Tukey’s multiple comparisons correction, P value < 0.05) is shown. (c) Representative immunoblot analysis against MBP, HA-tag, and myc-tag.

3.4. Confirming Protein Expression Level by Immunoblotting

It is important to ensure that expression levels of both AraC and competitor AraC* fusions are similar to have an accurate measure of homodimer versus heterodimer formation. Expression levels of AraC and AraC* fusions can be detected by immunoblotting against the HA-tag and myc-tag, respectively. Additionally, comparing expression levels for co-expression of both AraC and AraC* chimera versus expression of either AraC or AraC* chimera alone can be done by blotting against MBP.

1. Using the remaining samples from the 24-well deep well plate (see Note 16), take a representative culture for each construct and dilute the samples to OD₆₀₀ = 0.1 (final volume 20 µL) in a 1.5 mL Eppendorf tube.

2. Add 10 µL of 8M urea and 10 µL of 4x SDS loading dye.

3. Boil samples at 95°C for 5 minutes.

4. Load 10 µL of each sample onto an 8% SDS-Polyacrylamide gel. Store remaining samples at −80ºC (see Note 17).

5. Separate proteins by running the gel at 130V using the SDS-PAGE running buffer.

6. Electro-transfer the separated proteins from the gel onto a nitrocellulose membrane at 100V for 1 hour with an electroblotting apparatus at 4 ºC using the Transfer buffer.

7. After transfer, incubate the membrane with 5% BSA in 1x TBS-T to block for 1 hour at room temperature with continuous rocking.

8. Wash the membrane 3 times for 5 minutes with 1x TBS-T while rocking.

9. Incubate membrane with anti-MBP HRP-conjugated primary antibody (1:10000 dilution) in 1x TBS overnight at 4°C (or at room temperature for 1 hour).

10. Remove the primary antibody solution (see Note 18).

11. Wash the membrane 5 times with 1x TBS-T.

12. Incubate the membrane with the chemiluminescent HRP substrate following the manufacturer’s guidelines.

13. Image membrane (Figure 3c).

14. Determine the relative difference in expression level between fusions (if any) using an image analysis software (see Notes 19 and 20).

3.5. Confirming Chimera Insertion Topology by Maltose Complementation Assay

Proper integration of the fusion into the inner membrane can be confirmed by growing MBP-deficient E. coli strain (MM39) transformed with either an AraC or AraC* construct (no co-transformation) on media containing maltose as the sole carbon source.

1. Make M9 minimal media plates: Mix 1x M9 Minimal Salts with 1.5% (w/v) agar, and autoclave. Once the media has cooled to ~60°C, mix the agar and M9 components, add 0.4% (w/v) maltose, 1 mM MgSO₄, 0.05 mM CaCl₂, and the appropriate antibiotic (100 µg/mL Amp, 50 µg/mL Kan) (see Note 21).

2. Each AraC and AraC* construct of interest is transformed into MM39 cells and streaked onto selective LB plates.

3. After overnight incubation, individual colonies are picked and grown in selective LB media.

4. Each culture is individually streaked onto selective M9 minimal medium plates and incubated for 3 days at 37 ºC (Figure 4) (see Note 22).

Figure 4. Maltose complementation test.

The indicated pAraTMwt or pAraTMDN constructs were transformed into the MBP-deficient E. coli strain MM39. Saturated culture from each chimera was streaked onto M9 minimal media plates containing maltose. No growth was observed in the empty vectors, in which no AraC or AraC* fusion is expressed. Recovery of growth was observed for all other fusion constructs, indicating proper orientation in the cell membrane.

3.6. Confirming Chimera Insertion Topology by Spheroplast Protection Assay

Proper membrane integration and orientation can be also confirmed by specifically probing for the localization of MBP using a modified spheroplast digestion assay.

1. Inoculate 1 mL of LB media with a single MM39 colony transformed with the appropriate AraC or AraC* constructs (see 3.5).

2. Grow at 37ºC until OD₆₀₀ ~ 0.6, making sure the culture stays in the log phase or early stationary phase before treating.

3. Pellet the entire culture by centrifugation.

4. Resuspend the pellet with 50 µL of Periplasting buffer until the cell suspension solution is homogenous (see Note 23)

5. Incubate for 5 minutes at room temperature.

6. Add 50 µL of cold (4 ºC) ultra-pure water and mix by inverting.

7. Incubate for 5 minutes on ice.

8. Centrifuging (12000 x g) for 5 minutes.

9. Transfer the supernatant (Periplasmic Fraction; PF) into a clean tube and store it at 4ºC for further analysis.

10. Resuspend the pellet with 100 µL of lysis buffer (containing OmniCleave 400 U/mL).

11. Add 1 µL of 1.0 M MgCl₂ solution and mix by inverting.

12. Incubate for 5 minutes at room temperature.

13. Centrifuge (12000 x g) for 5 minutes.

14. Transfer the supernatant (Spheroplastic Fraction; SF) into a clean tube.

15. Add 2 µL of 500 mM EDTA solution (to chelate the residual magnesium) and mix by inverting.

16. Split the Spheroplastic Fraction into four equal samples.

17. To the first sample, add: proteinase K (0.002U/µL) – to release MBP from the inner membrane (SF1).

18. To the second sample, add: NP-40 alone (0.1% final) - to dissolve the spheroplasts and release the cytosolic proteins (SF2).

19. To the third sample, add: proteinase K (0.002U/µL) and NP-40 (0.1% final) (SF3).

20. The fourth sample is untreated control (SF4).

21. Detect the presence of MBP in the Periplasmic and Spheroplastic (SF1, SF2, SF3, and SF4) fractions by immunoblotting as described above.

4. Notes

1. The DNA sequence coding for the receptor domains of interest can be subcloned into both the pAraTMwt and pAraTMDN plasmids using ScaI and KpnI by either amplifying their coding sequence from template DNA sequences (e.g., cDNA coding for the full-length receptors) or directly using synthesized gene fragments from commercial sources. In both cases, ensure that SacI and KpnI sites are not already present in the sequence of the domain of interest.

2. The receptor domains of interest must include at least the membrane-spanning TM residues (typically around 20 residues long). The TM residues can be identified using UniProt (https://www.uniprot.org/) or web tools such as Membrane Protein Explorer (MPEx; https://blanco.biomol.uci.edu/mpex/) and TM Finder (http://tmfinder.research.sickkids.ca/cgi-bin/TMFinderForm.cgi). However, the DN-AraTM assay allows for the subcloning and expression of longer receptor domains, including juxta-membrane regions. We have cloned and successfully tested constructs with 40 extracellular juxtamembrane residues, the TM domain, and 40 cytosolic juxtamembrane residues (40TM40). While 5TM20 constructs have typically shown the most consistent results with the domains we have tested so far, optimization may be necessary.

3. Proper subcloning can be confirmed by sequencing using the common oligonucleotide primer: 5’-GCCGTGCGTACTGCGCTCAT-3’ (AraC-seq). If needed, subcloning in pAraTMwt can be confirmed using the 5’-ATGATGTGCCGGATTATGC-3’ (HA tag), and in pAraTMDN using 5’-CTGATTAGCGAAGAAGATCTGGA-5’ (Myc tag).

4. pAraTMwt and pAraTMDN carry the Ampicillin and Kanamycin resistance genes, respectively.

5. The assay can be scaled up using 48-well deep well plates (5 mL), which we routinely do.

6. Mix in glycerol by simply inverting the tube, and slowly add in the SDS afterward to ensure thorough mixing and resuspension.

7. If PCR amplification is unsuccessful, setting up a gradient PCR can simultaneously test multiple annealing temperatures, which may increase the efficacy of the reaction systematically. The smaller the size of the insert, the more difficult it can be to see a resolvable band upon gel extraction. If the efficiency of digests is subpar, doing sequential PCR reactions with the initial PCR product can help to obtain a larger yield.

8. With amplicons/digestion products that are close and small in size (typically 100–200bp), using a higher percentage of agarose when gel purifying can help to resolve small bands (1–2% is recommended).

9. The transmembrane domain insert sizes tend to be very small in comparison to the vector size (100–200 bp versus ~6000 bp). Therefore, the ratio for a successful ligation will most likely require a higher ratio of insert:vector. Typically, a 3:1 ratio is recommended to start, but with such small insert ratios, starting with a ratio of 10:1 may be more suitable for most applications.

10. When studying heterodimerization, samples 3 and 6 (Table 1) are negative controls because they should only result in a minimal GFP signal. A comparison of the results obtained with samples 2 and 7 indicates whether homodimer or heterodimer is favored. Comparing samples 5 and 8 should lead to a similar result. Finally, point mutations can be introduced in either AraC and AraC* construct. However, studying the effect of mutations on homodimerization does not require co-transformation with AraC* constructs (Table 2), as the GFP signal obtained with the wildtype and mutations can be directly compared.

11. While co-transformation of E.coli SB1676 cells is possible using heat-shock transformation, higher transformation efficiency can be obtained by electroporation using homemade electrocompetent cells. Moreover, it is recommended to prepare a stock of SB1676 competent cells already transformed with the pAraGFPCDF reporter plasmid for higher efficiency.

12. It is desirable to obtain many resolvable colonies on a given plate.

13. At least 3 individual colonies from each transformation are needed for each assay. In addition, 3 wells should contain LB media alone.

14. Growth time should be optimized, as it is important not to overgrow the cultures to prevent entering bacteria stationary phase and death, thereby reducing the efficacy of the assay. We recommend no more than 16–18 hours.

15. Leave only 100 µL in the wells corresponding to the 1:8 dilutions to ensure that every well contains the same volume.

16. Keep the remaining undiluted culture from the 24-well deep well plate at 4ºC for immunoblotting.

17. Samples tend to be very viscous because of the addition of urea, it is beneficial to load samples onto the gel before they fully cool down (not hot, slightly warm is best).

18. If the primary antibody is not HRP-conjugated, wash the membrane 3 times for 5 minutes with 1x TBS-T, and incubate with an appropriate HRP-conjugated secondary antibody in 1x TBS-T for 1 hour at room temperature.

19. If the juxtamembrane domains of the AraC and AraC* fusions differ in length, co-expression of these fusions, followed by immunoblotting against MBP, may result in discernable bands.

20. If small differences in expression levels (< 5%) are measured between fusion, these differences can be used to normalize the GFP emission signals.

21. Two sets of M9 plates must be prepared: One to test proper insertion of AraC-based fusions (AmpR), and another for AraC*-based fusions (KanR).

22. If no growth is observed, it indicates that the fusion of interest is either improperly inserted or not expressed.

23. Excessive agitation may cause premature lysing of the spheroplasts and can lead to contamination of the periplasmic fraction with cytoplasmic proteins.