Application of mRNA arrays for the production of mCherry reporter-protein arrays for quantitative gene expression analysis

DNase and one C F mplete protease inhibitor (Roche). Cells were lysed by brief homogenization followed by sonication. The lysate was clarified by centrifugation at 40,000 g at 4°C for 30 minutes and loaded onto a HisTrap FF column (GE Healthcare) equilibrated in lysis/binding buffer using an ÄKTA Purifier. His 6 -SpyCatcher was eluted using a 10 column-volume gradient to 100% elution buffer (20 mM Na 2 HPO 4 pH 7.4, 0.5 M NaCl, 500 mM imidazole). The N-terminal hexahistidine tag was removed by TEV cleavage (1 U TEV per 1 µg His 6 -SpyCatcher) at room temperature for 15 hours using the ProTEV Plus Kit (Promega). SpyCatcher was concentrated to a final volume of 0.5 ml using a Vivaspin centrifugal concentrator with a MWCO of 10 kDa (Sartorius). Concentrated SpyCatcher was loaded onto a HiLoad 16/600 Superdex 75 prep grade size exclusion column (GE Healthcare) equilibrated in 1X PBS using an ÄKTA Purifier. SpyCatcher was eluted in 1X PBS and aliquots were stored at -70 ° C.


Contents
. DNA sequences of plasmids generated during this study p9 Table S2. Primers for overlap extension PCR p10 Table S3. DNA sequences of in vitro transcription templates used during this study p12 Figure S1. The SpyCatcher/SpyTag protein coupling system p13 Figure S2. Gene expression analysis of mCherry-SpyTagsa p14 Figure S3. Schematic of the mechanism of post-transcriptional gene expression regulation by toehold switches p15 Figure S4. Linear correlation between DNA intensity and spotted DNA concentration for toehold switch-mCherry-SpyTagsa templates.
p17 Figure S6. Mounting of the in vitro transcription/translation slide sandwiches onto the slide separator assembly.

Preparation of DNA and mRNA capture surfaces
Two NHS-activated microarray slides (Nexterion H, Schott) were assembled in a sandwich arrangement so that the activated surfaces faced each other. 30 µl of either 1 mg/ml or 2 mg/ml streptavidin, in phosphate buffered saline pH 7.4 (PBS), were pipetted between the slides, as filling, to produce the DNA and mRNA capture surfaces, respectively. The assembly was incubated in a humidity chamber at 37°C for 30 minutes. To block any unreacted NHS functional groups, the slides were incubated in 50 ml 50 mM ethanolamine pH 8.5 in PBST (PBS containing 0.02% (v/v) Tween 20) at room temperature, with rocking, for 20 minutes. A standard wash and dry step was performed as follows: a wash with 50 ml PBST at room temperature, with rocking, for 5 minutes followed by a wash with 50 ml MilliQ H2O at room temperature, with rocking, for 30 seconds and a final rinse with MilliQ H2O. The slides were placed in a 50 ml Falcon tube and dried by centrifugation at 180 g, at room temperature for 30 seconds.
This was cloned into the BglII and XhoI sites of pET28a (Merck) to generate plasmid pET28-SpyTagsa.
Sequence encoding mCherry was amplified from p3-mCherry (a kind gift from the European Xenopus Resource Centre) using forward primer 5'-CGA GGC CAT ATG ATG GTG AGC AAG GGC GAG GA-3' and reverse primer 5'-GCTC CGG CTA GCC TTG TAC AGC TCG TCC ATG CCG-3'. This was cloned into the NdeI and NheI sites of pET28-SpyTagsa to generate pET28-mCherry-SpyTagsa (Table S1). A series of pET28-toehold X-mCherry-SpyTagsa toehold switch plasmids, based on the second-generation toehold switches 1, 3, 8 and 9 described in Green et al., 2014 13 , were generated by homologous recombination 30,31 . Briefly, linear pET28-mCherry-SpyTagsa plasmid was generated using pET28-mCherry-SpyTagsa as the template, forward primer 5'-CAC CAC CAC CAC CAC CAT ATG ATG GTG AGC-3' and reverse primer 5'-AGA TCT CGA TCC TCT ACG CCG GAC GCA TC-3'. Toehold switches, flanked at the 5' end by sequence with homology to pET28 and the T7 promoter, and at the 3' end by sequence with homology to His6-mCherry, were generated by overlap extension PCR (see Table S2 for primer sequences). PCR products were purified using Nanosep centrifugal filter columns with a 10 kDa MWCO (Pall Corporation). Escherichia coli DH5a cells were transformed with approximately 5 fmol of a mixture of a 1:10 molar ratio of linearized plasmid:toehold switch PCR product.

Preparation of DNA template arrays
Between 1.75 nM and 224 nM (see Figure legends for details) of 5' biotinylated, DNA in vitro transcription template in PBS was spotted onto a streptavidin-coated DNA capture surface using an automated arrayer (Genetix Qarray 2). 4 x 4 fields were spotted using a 175 µm pin head, allowing for a spot separation of 1125 µm. Spotted slides were incubated in a humidity chamber at 37°C for 20 minutes then washed and dried using a standard wash and dry step performed as follows: a wash with 50 ml PBST (PBS containing 0.02% (v/v) Tween 20) at room temperature, with rocking, for 5 minutes followed by a wash with 50 ml MilliQ H2O at room temperature, with rocking, for 30 seconds and a final rinse with MilliQ H2O. The slides were placed in a 50 ml Falcon tube and dried by centrifugation at 180 g, at room temperature for 30 seconds.

Generation of mRNA arrays
A DNA template array and streptavidin-coated RNA capture slide were mounted onto the slide separator device described in Figure  The slides were separated in MilliQ H2O and the newly generated mRNA array was washed and dried using the standard wash and dry step.

SpyCatcher protein expression and purification
The His6-SpyCatcher expression plasmid, pDEST14-SpyCatcher, was obtained from Prof. Mark Howarth (University of Oxford, UK). E. coli BL21(DE3) transformed with pDEST14-SpyCatcher was grown in 0.5 L LB supplemented with 100 µg/ml ampicillin at 37°C, with shaking, until the culture reached an OD600 of 0.5. Isopropyl b-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and the culture was incubated at 37°C, with shaking, for a further 3 hours.
Cells were harvested by centrifugation at 12,000 g, at 4°C for 30 minutes and resuspended in 50ml of lysis/binding buffer (20 mM Na2HPO4 pH 7.4, 0.5 M NaCl, 50 mM imidazole) supplemented with 1 mg/ml lysozyme, 1 U/ml DNase I and one CFmplete protease inhibitor tablet (Roche). Cells were lysed by brief homogenization followed by sonication. The lysate was clarified by centrifugation at 40,000 g at 4°C for 30 minutes and loaded onto a HisTrap FF column (GE Healthcare) equilibrated in lysis/binding buffer using an ÄKTA Purifier. His6-SpyCatcher was eluted using a 10 column-volume gradient to 100% elution buffer (20 mM Na2HPO4 pH 7.4, 0.5 M NaCl, 500 mM imidazole PBS and aliquots were stored at -70°C.

mCherry-SpyTag protein expression and purification
mCherry-SpyTag was expressed from pET28-mCherry-SpyTagsa and purified essentially as described for SpyCatcher.   Table S2. Primers for overlap extension PCR. Sequences corresponding to the toehold switches are shown in grey and flanking sequences, with homology to pET28-mCherry-SpyTagsa, are shown in blue. Sequences corresponding to the triggers are shown in red.

Construct
Primer Sequences Toehold switch 1 PCR product    Toehold switches consist of a toehold sequence (red) upstream of sequence that is capable of forming a hairpin structure and contains the ribosome binding site (RBS; blue), start codon (ATG; purple) and a linker sequence (lilac). They are inserted in-frame, upstream of the coding sequence for the protein of interest (mCherry-SpyTag; green). In the translation "off" state, a hairpin forms that sequesters the ribosome binding site and start codon. In the translation "on" state, a trans-acting, cognate, trigger RNA (cyan) binds the toehold sequence, completes branch migration with the hairpin and releases the ribosome binding site and start codon. Figure S4. Linear correlation between DNA intensity and spotted DNA concentration for toehold switch-mCherry-SpyTagsa templates. Plots of DNA intensity against spotted DNA concentration for the DNA template arrays generated and used in the experiment described in Figure 3. Figure S5. Linear correlation between RNA intensity and DNA intensity for toehold switch-mCherry-SpyTagsa mRNAs. Plots of RNA intensity against DNA intensity for the mRNA arrays generated and used in the experiment described in Figure 3. (B) Following in vitro transcription/translation, the slide separator assembly is lowered into a reservoir of MilliQ H2O in such a way that the flat steel bar prevents the entire assembly from sinking. Water ingress, promoted by the gravitational pull of the weight, decreases the surface tension inside the slide sandwich, separating the template and capture slides.