Reporter Gene Repression Assay to Study Translational Regulation of a Target Gene

Overview

This video demonstrates an in vivo assay in bacteria to study the interaction of RNA-binding proteins (RBPs) with RNA. The bacterial cells are transformed with two plasmid constructs — a binding-site plasmid encoding an mRNA containing a fluorescent reporter gene downstream of an RBP-binding site — while an RBP plasmid expresses the RBP proteins under the control of an inducer. Upon inducer-mediated increase in RBP production, the RBPs bind to the mRNA, resulting in translational repression of the reporter via inhibition of ribosome binding.

Protocol

1. System Preparation

1. Design of binding-site plasmids
   1. Design the binding site cassette as depicted in Figure 1. Each minigene contains the following parts (5' to 3'): Eagl restriction site, ∼40 bases of the 5' end of the kanamycin (Kan) resistance gene, pLac-Ara promoter, ribosome binding site (RBS), AUG of the mCherry gene, a spacer (δ), an RBP binding site, 80 bases of the 5' end of the mCherry gene, and an ApaLI restriction site.
      NOTE: To increase the success rate of the assay, design three binding-site cassettes for each binding site, with spacers consisting of at least one, two, and three bases. See the Representative Results section for further guidelines.
2. Cloning of binding site plasmids
   1. Order the binding-site cassettes as double-stranded DNA (dsDNA) minigenes. Each minigene is ∼500 bp long and contains an Eagl restriction site and an ApaLI restriction site at the 5' and 3' ends, respectively (see step 1.1.1).
      NOTE: In this experiment, mini-genes with half of the kanamycin gene were ordered to facilitate screening for positive colonies. However, Gibson assembly is also suitable here, in which case the binding site can be ordered as two shorter complementary single-stranded DNA oligos.
   2. Double-digest both the mini-genes and the target vector with Eagl-HF and ApaLI by the restriction protocol, and column purify.
   3. Ligate the digested minigenes to the binding-site backbone containing the rest of the mCherry reporter gene, terminator, and a kanamycin resistance gene.
   4. Transform the ligation solution into Escherichia coli TOP10 cells.
   5. Identify positive transformants via Sanger sequencing.
      1. Design a primer 100 bases upstream to the region of interest (see Table 1 for primer sequences).
      2. Miniprep a few bacterial colonies.
      3. Prepare 5 µL of a 5 mM solution of the primer and 10 µL of the DNA at 80 ng/µL concentration.
      4. Send the two solutions to a convenient facility for Sanger sequencing.
   6. Store purified plasmids at -20 °C, and bacterial strains as glycerol stocks, both in the 96-well format. DNA will then be used for transformation into E. coli TOP10 cells containing one of four fusion-RBP plasmids (see step 1.3.5).
3. Design and construction of the RBP plasmid
   NOTE: Amino acid and nucleotide sequences of the coat proteins used in this study are listed in Table 2.
   1. Order the required RBP sequence lacking a stop codon as a custom-ordered dsDNA minigene lacking a stop codon with restriction sites at the ends (Figure 1).
   2. Clone the tested RBP lacking a stop codon immediately downstream of an inducible promoter and upstream of a fluorescent protein lacking a start codon (Figure 1), similar to steps 1.2.2-1.2.4. Make sure that the RBP plasmid contains a different antibiotic resistance gene than the binding-site plasmid.
   3. Identify positive transformants via Sanger sequencing, similar to step 1.2.5 (see Table 1 for primer sequences).
   4. Choose one positive transformant and make it chemically competent. Store as glycerol purified plasmids at -20 °C and glycerol stocks of bacterial strains at -80 °C in 96-well plates.
   5. Transform the binding-site plasmids (from step 1.2.6) stored in 96-well plates into chemically-competent bacterial cells already containing an RBP-mCerulean plasmid. To save time, instead of plating the cells on Petri dishes, plate them using an 8-channel pipettor on 8-lane plates containing Luria-Bertani (LB) agar with relevant antibiotics (Kan and Amp). Colonies should appear in 16 h.
   6. Select a single colony for each double transformant and grow overnight in LB medium with the relevant antibiotics (Kan and Amp) and store as glycerol stocks at -80 °C in 96-well plates.

2. Experiment Setup

NOTE: The protocol presented here was performed using a liquid-handling robotic system in combination with an incubator and a plate reader. Each measurement was carried out for 24 inducer concentrations, with two duplicates for each strain + inducer combination. Using this robotic system, data for 16 strains per day with 24 inducer concentrations was collected. However, if such a device is unavailable, or if fewer experiments are necessary, these can easily be done by hand using an 8-channel multi-pipette and adapting the protocol accordingly. For example, preliminary results for four strains per day with 12 inducer concentrations and four time points were acquired in this manner.

1. Prepare, in advance, 1 L of bioassay buffer (BA) by mixing 0.5 g of tryptone, 0.3 mL of glycerol, 5.8 g of NaCl, 50 mL of 1 M MgSO₄, 1 mL of 10x phosphate-buffered saline (PBS) buffer pH 7.4, and 950 mL of double distilled water (DDW). Autoclave or sterile filter the BA buffer.
2. Grow the double-transformant strains at 37 °C and 250 rpm shaking in 1.5 mL LB with appropriate antibiotics (kanamycin at a final concentration of 25 μg/mL and ampicillin at a final concentration of 100 μg/mL), in 48-well plates, over a period of 18 h (overnight).
3. In the morning, make the following preparations.
   1. Inducer plate. In a clean 96-well plate, prepare wells with semi-poor medium (SPM) consisting of 95% BA and 5% LB26 in the incubator at 37 °C. The number of wells corresponds to the desired number of inducer concentrations. Add C4-HSL to the wells in the inducer plate that will contain the highest inducer concentration (218 nM).
   2. Program the robot to serially dilute medium from each of the highest-concentration wells into 23 lower concentrations ranging from 0 to 218 nM. The volume of each inducer dilution should be sufficient for all strains (including duplicates).
   3. While the inducer dilutions are being prepared, warm 180 μL of SPM in the incubator at 37 °C, in 96-well plates.
   4. Dilute the overnight strains from step 2.2 by a factor of 100 by serial dilutions: first, dilute by a factor of 10 by mixing 100 μL of bacteria with 900 μL of SPM in 48-well plates, and then dilute again by a factor of 10 by taking 20 μL from the diluted solution into 180 μL of pre-warmed SPM, in 96-well plates suitable for fluorescent measurements.
   5. Add the diluted inducer from the inducer plate to the 96-well plates with the diluted strains according to the final concentrations.
4. Shake the 96-well plates at 37 °C for 6 h, while taking measurements of optical density at 595 nm (OD595), mCherry (560 nm/612 nm), and mCerulean (460 nm/510 nm) fluorescence via a plate reader every 30 min. For normalization purposes, measure the growth of SMP with no cells added.

Representative Results

Figure 1: Overview of system design and cloning steps. Illustration of the cassette design for the binding site plasmid (left) and RBP-mCerulean plasmid (right). The next step is consecutive transformations of both plasmids into competent E. coli cells, with RBP plasmids first. Double-transformants are then tested for their mCherry expression levels in increasing inducer concentrations; if the RBP binds to the binding site, mCherry levels decline as a function of mCerulean (gray bubble).

Table 1: Binding sites and sequencing primers. Sequences for the binding sites and binding site cassettes used in this study, as well as the primers for the sequencing reactions detailed in the protocol (steps 1.2.5.1 and 1.3.3)

Name	Binding site location, A in AUG = 1	Binding site sequence (RBS for controls)	Site: ATG to second mCherry codon GTGControls: RBS to second mCherry codon GTG	Source
MS2_wt_d5	5	acatgaggattacccatgt	atgcacatgaggattacccatgtcgtg	Gen9 Inc.
MS2_wt_d6	6	acatgaggattacccatgt	atggcacatgaggattacccatgtgtg	Gen9 Inc.
MS2_wt_d8	8	acatgaggattacccatgt	atggcgcacatgaggattacccatgtcgtg	Gen9 Inc.
MS2_wt_d9	9	acatgaggattacccatgt	atggcgccacatgaggattacccatgtgtg	Gen9 Inc.
MS2_U(-5)C_d8	8	acatgaggatcacccatgt	atgcacatgaggatcacccatgtggtg	Gen9 Inc.
MS2_U(-5)C_d9	9	acatgaggatcacccatgt	atggcacatgaggatcacccatgtgtg	Gen9 Inc.
MS2_U(-5)C_d8	8	acatgaggatgacccatgt	atgcacatgaggatgacccatgtggtg	Gen9 Inc.
MS2_U(-5)G_d9	9	acatgaggatgacccatgt	atggcacatgaggatgacccatgtgtg	Gen9 Inc.
MS2_struct_d9	9	cacaagaggttcacttatg	atggccacaagaggttcacttatggtg	Gen9 Inc.
MS2_struct_d8	8	cacaagaggttcacttatg	atgccacaagaggttcacttatgggtg	Gen9 Inc.
PP7wt_d5'	5	taaggagtttatatggaaaccctta	atgctaaggagtttatatggaaacccttacgtg	Gen9 Inc.
PP7wt_d6'	6	taaggagtttatatggaaaccctta	atgaataaggagtttatatggaaacccttagtg	Twist Bioscience
PP7wt_d8'	8	taaggagtttatatggaaaccctta	atgaacataaggagtttatatggaaacccttacgtg	Twist Bioscience
PP7wt_d9'	9	taaggagtttatatggaaaccctta	atgaacaataaggagtttatatggaaacccttagtg	Twist Bioscience
PP7_USLSBm_d6	6	taaccgctttatatggaaagggtta	atggctaaccgctttatatggaaagggttagtg	Gen9 Inc.
PP7_USLSBm_d15	15	taaccgctttatatggaaagggtta	atgggcgccggcgctaaccgctttatatggaaagggttagtg	Gen9 Inc.
PP7_nB_d5	5	taagggtttatatggaaaccctta	atgctaagggtttatatggaaacccttagcgtg	Gen9 Inc.
PP7_nB_d6	6	taagggtttatatggaaaccctta	atggctaagggtttatatggaaacccttatgtg	Gen9 Inc.
PP7_USs_d5	5	taaggagttatatggaaccctta	atgctaaggagttatatggaacccttagtg	Gen9 Inc.
PP7_USs_d6	6	taaggagttatatggaaccctta	atggctaaggagttatatggaacccttagcgtg	Gen9 Inc.
No_BS_d1	-	-	ttaaagaggagaaaggtacccatggtg	Gen9 Inc.
No_BS_d4	-	-	ttaaagaggagaaaggtacccatgggcgtg	Gen9 Inc.
No_BS_d10	-	-	ttaaagaggagaaaggtacccatgggcgccggcgtg	Gen9 Inc.
Sequencing primer for binding site cassettes			gcatttttatccataagattagcgg	IDT
Sequencing primer for RBP cassettes			gcggcgctgggtctcatctaataa	IDT

Table 2: RBP sequences. Amino acid and nucleotide sequences of the coat proteins used in this study

RBP name in this work	source organism name, protein	source organism gene	source organism refseq	wt aa seq	changes from wt (and references)	aa seq used in this work	nt seq used in this work
MCP	Escherichia virus MS2	cp	NC_001417.2	MASNFTQFVLV DNGGTGDVTV APSNFANGVA EWISSNSRSQ AYKVTCSVRQ SSAQNRKYTI KVEVPKVATQT VGGVELPVA AWRSYLNMEL TIPIFATNSD CELIVKAMQG LLKDGNPIPS AIAANSGIY	delF-G [1] V29I [1] taken from addgene plasmid 27121	MASNFTQFVLV DNGGTGDVTV APSNFANGIA EWISSNSRSQ AYKVTCSVRQ SSAQNRKYTI KVEVPKG AWRSYLNMEL TIPIFATNSD CELIVKAMQG LLKDGNPIPS AIAANSGIY	ATGGCTTCTA ACTTTACTCA GTTCGTTCTC GTCGACAATG GCGGAACTGG CGACGTGACT GTCGCCCCAA GCAACTTCGC TAACGGGATC GCTGAATGGA TCAGCTCTAA CTCGCGTTCA CAGGCTTACA AAGTAACCTG TAGCGTTCGT CAGAGCTCTG CGCAGAATCG CAAATACACC ATCAAAGTCG AGGTGCCTAA AGGCGCCTGG CGTTCGTACT TAAATATGGA ACTAACCATT CCAATTTTCG CCACGAATTC CGACTGCGAG CTTATTGTTA AGGCAATGCA AGGTCTCCTA AAAGATGGAA ACCCGATTCC CTCAGCAATC GCAGCAAACT CCGGCATCTAC
PCP	Pseudomonas phage PP7	cp	NC_001628.1	MSKTIVLSVGEA TRTLTEIQST ADRQIFEEKV GPLVGRLRLT ASLRQNGAKT AYRVNLKLDQ ADVVDCSTSVC GELPKVRYTQ VWSHDVTIVA NSTEASRKSL YDLTKSLVAT SQVEDLVVNL VPLGR	delF-G [2] taken from addgene plasmid 40650	MLASKTIVLSVG EATRTLTEIQ STADRQIFEE KVGPLVGRLR LTASLRQNGA KTAYRVNLKL DQADVVDSG LPKVRYTQVW SHDVTIVANS TEASRKSLYD LTKSLVATSQ VEDLVVNLVP LGR	ATGCTAGCCTC CAAAACCATC GTTCTTTCGG TCGGCGAGGC TACTCGCACT CTGACTGAGA TCCAGTCCAC CGCAGACCGT CAGATCTTCG AAGAGAAGGT CGGGCCTCTG GTGGGTCGGC TGCGCCTCAC GGCTTCGCTC CGTCAAAACG GAGCCAAGAC CGCGTATCGC GTCAACCTAA AACTGGATCA GGCGGACGTC GTTGATTCCG GACTTCCGAA AGTGCGCTAC ACTCAGGTAT GGTCGCACGA CGTGACAATC GTTGCGAATA GCACCGAGGC CTCGCGCAAA TCGTTGTACG ATTTGACCAA GTCCCTCGTC GCGACCTCGC AGGTCGAAGA TCTTGTCGTC AACCTTGTGC CGCTGGGCCGT

Materials

Name	Company	Catalog Number	Comments
Ampicillin sodium salt	SIGMA	A9518
Magnesium sulfate (MgSO4)	ALFA AESAR	33337
48 plates	Axygen	P-5ML-48-C-S
8-lane plates	Axygen	RESMW8I
96-well plates	Axygen	P-DW-20-C
96-well plates for plate reader	Perkin Elmer	6005029
ApaLI	NEB	R0507
Binding site sequences	Gen9 Inc. and Twist Bioscience		see Table 1
E. coli TOP10 cells	Invitrogen	C404006
Eagl-HF	NEB	R3505
Glycerol	BIO LAB	71205
Incubator	TECAN	Liconic incubator
Kanamycin solfate	SIGMA	K4000
KpnI- HF	NEB	R0142
Ligase	NEB	B0202S
Liquid-handling robotic system	TECAN	EVO 100, MCA 96-channel
MATLAB analysis software	Mathworks
Multi-pipette 8 lanes	Axygen	BR703710
N-butanoyl-L-homoserine lactone (C4-HSL)	Cayman	K40982552 019
PBS buffer	Biological Industries	020235A
Plate reader	TECAN	Infinite F200 PRO
Q5 HotStart Polymerase	NEB	M0493
RBP sequences	Addgene	27121 & 40650	see Table 2
Sodium Chloride (NaCl)	BIO LAB	190305
SV Gel and PCR Clean-Up System	Promega	A9281
Tryptone	BD	211705