Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Summary

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Abstract

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Introduction

DNA is often referred to as the blueprint of a living cell since all structures of the cell are encoded in the sequence of its DNA. Accurate replication and DNA repair mechanisms ensure that only very low rates of mutations occur, which is essential for sustaining correct functions of coded genes. Changes of the DNA sequence can affect successive functions at different levels starting with DNA (recognition by transcription factors and restriction enzymes), then RNA (base-pair complementarity and secondary structure alterations) and/or protein (amino acid substitutions, deletions, additions or frame-shifts). While many mutations do not affect gene function significantly, some mutations in the DNA can have huge implications. Thus, site-directed mutagenesis is a valuable tool for studying the importance of specific DNA sites at all levels.

This protocol describes a targeted mutagenesis approach used to introduce specific mutations. The protocol relies on two different PCR strategies: a 2-step or a 3-step PCR. The 2-step PCR is applicable if the desired mutation is close to either the 5' end or the 3' end of the DNA of interest (<200 base pairs (bp) from the end) and the 3-step PCR is applicable in all cases.

In the 2-step PCR approach, 3 primers are designed, in which one set of primers is designed to amplify the DNA of interest (primers 1 and 3, forward and reverse, respectively), and a single primer is designed to incorporate the mutation. The mutation introducing primer (primer 2) should have a reverse orientation if the mutation is close to the 5' end and a forward orientation if the mutation is close to the 3' end. In the first PCR step, primer 1+2 or 2+3 amplifies a small fragment close to the 5' end or 3' end, respectively. The resulting PCR product is then used as a primer in step two with primer 1 or 3, thus resulting in a PCR product with a mutation in the DNA of interest (Figure 1A).

In the 3-step PCR, 4 primers are designed, in which one set of primers is designed to amplify the DNA of interest (primers 1 and 4, forward and reverse, respectively) and one set of primers is designed to incorporate specific mutations with overlapping complementarity (primers 2 and 3, reverse and forward, respectively). In step one and two, primers 1+2 and 3+4 amplify the 5' and 3' end. In step three, the resulting PCR products from step one and two are used as templates and amplified with primers 1+4. Thus, the resulting PCR product is the DNA of interest with the desired mutation (Figure 1B).

While the mutated DNA can be used for any downstream application, this protocol describes how to re-combine the DNA into a cloning vector. The use of cloning vectors has several advantages such as ease of cloning and specific experimental applications depending on features of the vector. This feature is often used for RNA interaction studies. Another technique for RNA interaction studies is structural probing of the RNA in complex with another RNA^1,2 or protein^3,4. However, structural probing is only performed in vitro whereas site-directed mutagenesis and subsequent cloning allow for interaction studies in vivo.

Site-directed mutagenesis has been extensively used for RNA interaction studies as presented here. However, the key method regarding 2- or 3-step PCR is applicable to any piece of DNA, and thus not only limited to RNA-interaction studies.

To exemplify the technique and its possible uses, characterization of regions important for post-transcriptional regulation of the mRNA, csgD, of Escherichia coli (E. coli) is used. In E. coli, csgD is targeted by the small non-coding RNA, McaS, in cooperation with a protein, Hfq, to repress protein-expression of CsgD^2,4,5. The technique is used to introduce mutations to the base-pairing region between csgD and McaS, and to the Hfq binding site of csgD. The obtained DNA is then cloned into a vector suitable for subsequent experiments. Downstream applications of the technique include both in vivo and in vitro experiments. For illustration, example 1 is characterized in vivo using a western blot assay and example 2 is characterized in vitro using an electrophoretic mobility shift assay (EMSA). In both cases, it is illustrated how site-directed mutagenesis can be used in combination with other techniques to make biological conclusions about a gene of interest.

Protocol

1. Vector selection

1. Choose a vector to perform downstream experiments with. Any vector is applicable for this 2- and 3-step PCR method.
2. Based on choice of vector, choose appropriate restriction enzymes for cloning.

2. Primer design for site directed mutagenesis

1. Decide between either the 2-step or 3-step PCR strategy (2-step is only for mutations <200 bp from either end of the DNA of interest). For the 2-step PCR, go to step 2.2 and for the 3-step PCR go to step 2.3.
2. Design primers for 2-step PCR.
   1. Design primer 1 and 3 to amplify the DNA of interest and with a 5’ overhang that contains 4 nucleotides (e.g., ATAT or AGCT) followed by the relevant restriction recognition site necessary to clone into the chosen vector.
   2. Design primer 2 to introduce mutation(s) at the desired site(s) and flank the mutation with 10-15 complementary nucleotides on both sides. Make the primer reverse if the mutation is introduced at the 5’ end or forward if the mutation is introduced at the 3’ end.
3. Design primers for 3-step PCR.
   1. Design primer 1 and 4 to amplify the DNA of interest and with a 5’ overhang that contains 4 nucleotides (e.g., ATAT or AGCT) followed by the relevant restriction recognition site necessary to clone into the chosen vector.
   2. Design primer 2 and 3 to introduce mutation(s) at desired site(s) and flank the mutation by 10-15 complementary nucleotides on both sides. Primer 2 and 3 are reverse complementary.

3. PCR amplification of wild type DNA for cloning

NOTE: For details on PCR, see⁶.

1. Perform PCR⁶ using primers 1+2 (2-step PCR) or 1+4 (3-step PCR) and use wild type DNA as template to obtain PCR product I. Use the PCR program in Table 1.
2. Validate PCR by agarose gel electrophoresis.
   1. Make an agarose gel solution (2%) by adding 2 g of agarose per 100 mL of 1x Tris-acetate-ethylenediaminetetraacetic acid (EDTA) (TAE) buffer. Dissolve the agarose by boiling in a microwave oven. Add the DNA-staining dye ethidium bromide (to a final concentration of ~ 0.5 µg/mL) to the agarose gel solution for visualization.
   2. Cast agarose gel, place it in an electrophoresis unit, and load PCR samples (mixed with DNA loading dye) and a DNA ladder of known size. Run samples at 75 W for 45 min, or until bands are separated adequately, and visualize bands at an ultra-violet (UV) table or with a gel imaging system.
3. Purify the PCR product with a gel extraction kit (Table of Materials) and measure the concentration of purified DNA with a spectrophotometer (Table of Materials).
4. Store the purified DNA at -20 °C (in Tris-EDTA (TE) buffer – long term storage) or 4 °C (in dH₂O – short term storage) until used in step 5.

4. PCR to introduce site-directed mutations in the DNA

1. PCR for 2-step PCR (see step 4.2 for 3-step PCR)
   1. Perform PCR⁶ using primers 1+2 if mutations are in the 5’ end or 2+3 if the mutations are in the 3’ end and use wild type DNA as a template to obtain PCR product II (see Table 1 for PCR program).
   2. Validate PCR by agarose gel electrophoresis as in step 3.2.1 and 3.2.2.
   3. Purify the PCR product with a gel extraction kit (Table of Materials) and measure the concentration of purified DNA with a spectrophotometer (Table of Materials).
   4. Store purified DNA at -20 °C (in TE buffer) or 4 °C (in dH₂O) until used in step 5.
   5. Perform PCR⁶ using PCR product II as the primer together with primer 3 if mutations are in the 5’ end or primer 1 if the mutations are in the 3’ end and use wild type DNA as template to obtain PCR product III (see Table 1 for PCR program).
   6. Validate PCR by agarose gel electrophoresis as in step 3.2.1 and 3.2.2.
   7. Purify PCR product with a gel extraction kit (Table of Materials) and measure concentration of purified DNA with a spectrophotometer (Table of Materials).
   8. Store purified DNA at -20 °C (in TE buffer) or 4 °C (in dH₂O) until step 5.
2. PCR for 3-step PCR
   1. Perform PCR⁶ using primers 1+2 and 3+4 in separate reactions and use wild type DNA as template to obtain PCR product II and III (see Table 1 for PCR program).
   2. Validate PCRs by agarose gel electrophoresis as in step 3.2.1 and 3.2.2.
   3. Purify PCR products with a gel extraction kit (Table of Materials) and measure the concentration of purified DNA with a spectrophotometer (Table of Materials).
   4. Store purified DNA at -20 °C (in TE buffer) or 4 °C (in dH₂O).
   5. Perform PCR⁶ using primers 1+4 and use 2-5 ng of both PCR products II and III as the templates (in the same reaction) to obtain PCR product IV (see Table 1 for PCR program).
   6. Validate PCR by agarose gel electrophoresis as in step 3.2.1 and 3.2.2.
      NOTE: It is not unusual to get several incorrect bands (can sometimes be reduced by using less template). However, the incorrect PCR products can be ignored if the correctly sized band is excised and gel-extracted.
   7. If correct, purify PCR product with a gel extraction kit (Table of Materials) and measure concentration of purified DNA with a spectrophotometer (Table of Materials).
   8. Store purified DNA at -20 °C (in TE buffer) or 4 °C (in dH₂O) until step 5.

5. Recombination of wild type and mutant version(s) of DNA into the chosen vector

NOTE: For details on following steps, see⁷.

1. Digest purified PCR products I and III (from 2-step PCR) and/or IV (from 3-step PCR) using the relevant restriction enzymes.
   1. In 20 µL of 1x digestion buffer with 1 µL of each restriction enzyme, digest 200 ng of PCR product at 37 °C for 30-60 min.
2. Purify digested PCR product by gel-extraction using a gel extraction kit (Table of Materials), measure the DNA concentration of purified DNA with a spectrophotometer (Table of Materials), and store at -20 °C (in TE buffer) or 4 °C (in dH₂O) until used for step 5.6.
3. Digest purified vector with relevant restriction enzymes and treat with alkaline phosphatase to decrease vector re-ligation events. Do not treat PCR products with alkaline phosphatase.
   1. In 30 µL of 1x digestion buffer with 1 µL of each restriction enzyme and 1 µL of alkaline phosphatase, digest 1,000 ng of the vector at 37 °C for 30-60 min.
4. Separate digested vector from waste DNA (e.g., uncut vector and cut-out DNA) using agarose gel electrophoresis as in step 3.2.1 and 3.2.2.
5. Purify digested vector by gel-extraction using a gel extraction kit (Table of Materials), measure DNA concentration of purified DNA with a spectrophotometer (Table of Materials), and store at -20 °C (in TE buffer) or 4 °C (in dH₂O) until used for step 5.6.
6. Ligate digested PCR products into digested vector with the reactions specified in Table 2.
7. Incubate at room temperature for 2 h or overnight at 16 °C.
8. Transform recipient strain (e.g., E. coli K12) with ligation reactions.
   1. Grow strain to OD₆₀₀=0.3-0.5 and transfer a 1 mL culture to as many 1.5 mL tubes as ligase reactions.
   2. Spin at 3,500 x g for 5 min and discard supernatant.
   3. Resuspend cells in 200 μL of transformation buffer (10 mL of lysogeny broth (LB) with 0.1 g/mL polyethylene glycol 3350, 5% dimethyl sulfate and 20 mM MgCl₂).
   4. Add ligation reaction, and place the tubes on ice for 30 min.
   5. Heat-shock for 2 min at 42 °C.
   6. Add 1 mL of LB to the 1.5 mL tubes, and allow phenotypic expression of antibiotic resistance for at least 45 min at 37 °C.
   7. Spin the cells at 3,500 x g for 5 min, discard 1 mL of supernatant, and resuspend the cells in the remaining supernatant.
   8. Plate the cells on plates with appropriate antibiotics and incubate overnight at 37 °C.
9. Identify transformants harboring vectors with successful integration of DNA insert (e.g., by PCR using vector- and insert-specific primers).
10. Validate sequence of DNA by sanger sequencing.
    CAUTION: Do not use the same primers for sequencing as used for step 5.9.

6. Using constructed vectors for in vitro and/or in vivo experiments

1. In vivo experiment
   NOTE: This is an example of using a vector to express wild type/mutated RNA to characterize post-transcriptional regulation. For further details on western blotting, see⁸.
   1. Grow E. coli K12 strains with constructed vectors in appropriate medium and induce expression if required. Harvest samples by centrifugation.
   2. Prepare samples for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) by dissolving cell pellets in 1x SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2.5% SDS, 0.002% bromophenol blue, 5% β-mercaptoethanol, 10% glycerol) and boil at 95 °C for 5 min.
      NOTE: It is possible to cast a gel or use a commercially available precast gel. The latter was used in the results presented in Figure 3 (Table of Materials).
   3. Load 10⁷ cells of each sample in separate wells (include a protein ladder), and run gel at 200 V until proteins are separated (approximately 45 min).
   4. Blot the proteins onto a cellulose-membrane by semi-dry transfer at 80 mA for 1 h.
   5. Block the membrane with a mixture of proteins (e.g., 5% milk-powder dissolved in 1x Tween 20-Tris-buffered saline (TTBS) buffer).
   6. Add primary antibody (dissolved in 1x TTBS buffer) that target the protein of interest (e.g., GFP-, FLAG-, or HIS-tagged protein) and incubate for 1 h with gentle agitation.
   7. Wash membrane in 1x TTBS for 10 min to remove unbound antibodies. Repeat twice more.
   8. Add secondary antibody (dissolved in 1x TTBS buffer) that targets the primary antibodies and allow for detection (e.g., horseradish peroxidase (HRP)-conjugated secondary antibodies. Incubate for 1 h with gentle agitation.
   9. Wash the membrane in 1x TTBS for 10 min to remove unbound antibodies. Repeat twice more.
   10. Visualize the membrane with a technique compatible with the secondary antibodies (e.g., by imaging after incubation with a luminol-derived chemiluminescence, if a HRP-conjugated secondary antibody was used).
2. In vitro experiment
   NOTE: This is an example of using the vector as a template for in vitro transcription of RNA to characterize RNA-protein interactions. For further details on EMSA, see⁹.
   1. Make in vitro transcripts using a T7 in vitro transcription kit (Table of Materials) and vectors from step 5 as templates.
   2. Separate RNA transcripts by PAGE on a 4.5% 7 M urea denaturing gel, and extract RNA directly from the gel by electro elution with dialysis tubes (Table of Materials).
   3. Label RNA (e.g., radiolabeling with γ-³²P-ATP using T4-polynucleotide kinase (Table of Materials) and purify again with columns (Table of Materials).
      CAUTION: Before working with radioactive material, consult with the local radiation safety officer.
   4. Mix labelled-RNA with increasing concentrations of protein in separate reactions in a 1x binding buffer (20 mM Tris, pH 8, 100 mM KCl, 1 mM MgCl₂, 1 mM dithiothreitol (DTT)).
      NOTE: In the presented results (Figure 4), 2 nM of radiolabeled csgD mRNA was mixed with a gradient of 0 to 2 µM Hfq protein (monomer concentration).
   5. Allow protein and RNA to hybridize before loading hybridization mix on a non-denaturing polyacrylamide gel and run the gel for 1.5 h at 200 V.
   6. Visualize the gel with a technique compatible with the labeling from step 6.1.3. (e.g., by phosphoimaging if radiolabeling was applied).
   7. Quantify the relative intensity of the shifted bands with an imaging processing program and fit a curve (sigmoidal) to the data by using a graph and data analysis software (Table of Materials). Based on the fitted curve, dissociation constant (K_d) values can be determined automatically with the software.

Results

To investigate RNA interactions regarding post-transcriptional regulation of csgD, a double vector setup was chosen: one to express the csgD mRNA and another to express the small non-coding RNA, McaS. csgD was cloned into pBAD33, which is an arabinose inducible medium-copy plasmid with chloramphenicol resistance and McaS was cloned into mini R1 pNDM220, which is an isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible low copy plasmid with ampicillin resis...

Discussion

Site-directed mutagenesis has a broad array of different applications, and here, representative results from an in vivo and an in vitro experiment were included as examples of how to make biological conclusions using the technique. Site-directed mutagenesis has for long been the golden standard for RNA interaction studies. The strength of the technique lies in the combination of introducing relevant mutations with downstream assays and experiments (e.g., western blot or EMSA) to draw conclusions about specific DNA sites ...

Materials

Name	Company	Catalog Number	Comments
Anti-GroEL antibody produced in rabbit	Merck	G6532	Primary antibody
Azure c200	Azure	NA	Gel imaging workstation
Custom DNA oligo	Merck	VC00021
DeNovix DS-11	DeNovix	NA	Spectrophotometer for nucleic acid measurements
DNA Gel Loading Dye (6X)	Thermo Scientific	R0611
Ethidium bromide solution 1 %	Carl Roth	2218.1
GeneJET Gel Extraction Kit	Thermo Scientific	K0691
GeneRuler DNA Ladder Mix	Fermentas	SM0333
Gerard GeBAflex-tube Midi	Gerard Biotech	TO12	Dialysis tubes for electro elution
MEGAscript T7 Transcription Kit	Invitrogen	AM1334
Mini-Sub Cell GT Cell	Bio-Rad	1704406	Horizontal electrophoresis system
Monoclonal ANTI-FLAG M2 antibody produced in mouse	Merck	F3165	Primary antibody
Mouse Immunoglobulins	Dako Cytomation	P0447	HRP conjucated secondary antibody
NucleoSpin miRNA	Macherey Nagel	740971	RNA purification
NuPAGE 4-12% Bis-Tris Protein Gels	Thermo Scientific	NP0323BOX	Bis-Tris gels for protein separation
Phusion High-Fidelity PCR Master Mix with HF Buffer	New England Biolabs	M0531S	DNA polymerase
PowerPac HC High-Current Power Supply	Bio-Rad	1645052
Rabbit Immunoglobulins	Dako Cytomation	P0448	HRP conjucated secondary antibody
SeaKem LE Agarose	Lonza	50004
SigmaPlot	Systat Software Inc	NA	Graph and data analysis software tool
T100 Thermal Cycler	Bio-Rad	1861096	PCR machine
T4 DNA ligase	New England Biolabs	M0202	Ligase
T4 Polynucleotide Kinase	New England Biolabs	M0201S
TAE Buffer (Tris-acetate-EDTA) (50X)	Thermo Scientific	B49