TRIAD: a transposition-based approach for gene mutagenesis by random short in-frame insertions and deletions for directed protein evolution

Insertions and deletions (InDels) are among the most frequent changes observed in natural protein evolution, yet their potential has hardly been harnessed in directed evolution experiments. Here we describe the standard protocol for TRIAD (Transposition-based Random Insertion And Deletion mutagenesis), a simple and e�cient Mu transposon mutagenesis approach for generating libraries of single InDel variants with one, two or three triplet nucleotide insertions or deletions. This method has recently been employed in three published examples of InDel-based directed evolution of proteins, including a phosphotriesterase, a scFv antibody and an ancestral luciferase.


Introduction
Access to reliable methods for gene randomization is essential for success in directed evolution experiments.Popular and practically useful methods must meet several key requirements: a good library generation protocol should create a large number of variants, avoid bias in gene composition or type of variant introduced, and be technically straightforward.When it comes to amino acid substitutions, several approaches (e.g.error-prone PCR, site-saturation mutagenesis starting with synthetic oligonucleotides) have been developed that partially or fully meet these criteria.In contrast, the use of insertions and deletions (InDels) in directed evolution experiments has been curtailed by practical limitations in existing methodologies to randomly incorporate such changes within a sequence of interest.Therefore, while the effects of substitutions are extensively documented in the literature, the application of InDels in protein engineering has been sparse, with very few directed evolution campaigns on record that originate from such libraries.For example, the random insertion/deletion (RID) mutagenesis protocol [1] , the rst attempt towards creating InDel libraries, relies on a complex protocol involving random cleavage of single stranded DNA, so that random substitutions are introduced unintentionally alongside the target mutations.Two other early methods, segmental mutagenesis [2] and Random insertional-deletional strand exchange mutagenesis (RAISE [3] ), do not control for the length of the InDel and consequently produce libraries that primarily contain frameshifted variants.In contrast, a codon-based protocol dubbed codonbased random deletion (COBARDE [4] ) gives a pool of multiple codon-based deletions with <5% frameshifts but requires custom reprogramming of an oligonucleotide synthesizer to create mutagenic oligonucleotides.Alternatively, the viability of transposon-based protocols has been established for generating deletions of various sizes, up to gene truncation variants [5] .However, the only reported such protocol to create insertions, namely pentapeptide scanning mutagenesis [6] , merely gains access to insertions of de ned size and sequence.
Here we present a set of procedures (dubbed TRIAD: Transposition-based Random Insertion And Deletion mutagenesis) [7] for random introduction of single short in-frame InDels of one, two or three nucleotide triplets (± 3, 6 or 9 bp) into a given DNA sequence.TRIAD consists of a single transposition reaction followed by successive cloning steps for the generation of deletions or insertions (Figure 1).TRIAD's rst step is an in vitro Mu transposition reaction [8] that ultimately determines the location of the forthcoming single InDel event in each variant.The reaction is performed using engineered mini-Mu transposons, dubbed TransDel (Figure 1A) and TransIns (Figure 1B), that are inserted randomly within the target DNA sequence during the rst step of TRIAD; resulting in the generation of transposon insertion libraries.The ends of TransDel and TransIns were designed to bring about deletion and insertion libraries, respectively.
TransDel is functionally equivalent to the previously described MuDel transposon [5] with recognition sites for the type IIS restriction enzyme MlyI at both ends.Taking advantage of the transposon insertion mechanism, the positioning of MlyI sites within TransDel enables the deletion of 3 bp at random positions within the target sequence upon MlyI digestion and self-ligation (Figure 2A), as previously described [5] .This strategy was extended to the generation of longer contiguous deletions (i.e., -6 and -9 bp) with a second stage, involving the insertion and subsequent MlyI-mediated removal of custom-made cassettes (dubbed Del2 and Del3; Figure 1A and Figure 2).For the generation of insertions, a new transposon, TransIns, was designed as -in contrast to TransDel -an asymmetric transposon (Figure 1B and Figure 2B), bearing different end sequences (NotI on one end and MlyI on the other).The latter site marks subsequent insertion sites for the ligation of custom-made shuttle cassettes: Ins1, Ins2 and Ins3 carrying one, two and three randomized nucleotide triplets, respectively.Further digestion using a type IIS restriction enzyme (AcuI) removes most of the shuttle sequence but leaves triplet insertions behind (Figure 1B and Figure 2B).
The use of TRIAD has, so far, been reported in three published examples of InDel-based directed evolution of proteins, including a phosphotriesterase [7] , a therapeutic scFv antibody [9] and, most recently, an ancestral luciferase [10] .

Procedure
Prior to applying this protocol, the gene of interest (GOI) needs to be inserted in a TRIAD-bespoke target vector such as pID-T7 or pID-Tet (See troubleshooting note 1).

Construction of transposon insertion libraries
This part describes how to generate transposon insertion libraries, i.e., with transposon (either TransDel or TransIns) incorporated only in the GOI and not elsewhere in the target vector.Use TransDel to construct deletion libraries and TransIns for insertion libraries.Mix up to 2.5 µl or up to 5 µl of puri ed transposition product with 25 µl or 50 µl of electrocompetent cells, respectively (see troubleshooting note 2).Set voltage to 1.8 kV when using 1 mm gap electroporation cuvettes (alternatively, use up to 2.5 kV when using 2 mm gap electroporation cuvettes).Add Recovery Medium (or, alternatively, use SOC medium) to a nal volume of 300 µl and incubate 1 hour under agitation at 37 °C.
Plate up to 250 µl of this transformed culture mix on LB-Agar supplemented with 100 µg/ml ampicillin and 34 µg/ml chloramphenicol (LB-Agar Amp + Cam) and incubate overnight at 37 °C.Alternatively, inoculate 10 mL liquid LB Amp + Cam with the transformed culture mix and incubate under agitation at 37 °C overnight.To assess transposition e ciency, plate 0.1 to 1% of transformed cells on LB-Agar Amp + Cam and, after overnight incubation at 37 °C, calculate the number of transformants present in the total transformation mixture from the number of counted colonies.
1.4 Extract plasmid library pool from E. coli transformants.
After growth, collect transformed E. coli cells and extract plasmid libraries.Depending on the conditions used for growth (solid or liquid growth): -If transformed cells were plated in a 14 cm Petri Dish, scrape colonies in 6 mL LB, transfer into three 2-ml Eppendorf tubes, pellet cells by centrifugation and extract plasmid pool from one tube (store the two others as back-ups) using a Midiprep kit.
-If transformed cells were grown in a 10 mL liquid culture, collect cell pellet by centrifugation and extract plasmid directly from the collected pellet using a Midiprep kit.
This step ultimately yields an intermediate plasmid library pool in which the transposons (either TransDel or TransIns) are incorporated at any viable insertion site in the target vector (i.e., the ampicillin resistance gene and the origin of replication are likely to be free of transposon at this stage).Separate the resulting fragments by agarose gel electrophoresis.The reaction will generate four products, corresponding to (in decreasing size): (i) target vector backbone with transposon, (ii) target vector without transposon, (iii) GOI with transposon and (iv) GOI without transposon.Isolate band corresponding to fragment (iii) and purify the DNA from the agarose gel.The target vector (band ii) may also be isolated and puri ed at this stage (alternatively, the digested vector can be prepared extemporaneously).
1.6 Ligate the GOI containing the transposon in the target vector.
Perform cohesive end ligation based on ratio 1 vs. 3 to 5 (vector/insert) in 20 µl reactions with T4 DNA ligase.Digested vector amount should be between 10 and 50 ng (calculate insert quantity accordingly).
Reaction ( nal volume: 20 µl): To estimate the transposon insertion library size, plate 0.1 to 0.5% of transformed cells on LB-Agar Amp + Cam and extrapolate the number of transformants present in the total transformation mixture from the number of resulting colonies.
1.8 After growth, collect cells and extract plasmid pool corresponding to transposon insertion library.Follow procedure described in step 1.4.

Construction of deletion libraries from TransDel insertion libraries
2.1 Construction of single nucleotide triplet deletion libraries (as outlined by Jones [5] ) Incubate 30 minutes at room temperature.Purify and concentrate ligation product in 10 µl nuclease-free water.Plate up to 250 µl of this transformed culture mix on LB-Agar Amp (100 µg/ml) and incubate overnight at 37 °C.Alternatively, inoculate 10 mL liquid LB Amp with the transformed culture mix and incubate overnight under agitation at 37 °C.To estimate library size, plate 0.1 to 0.5% of transformed cells on LB-Agar Amp and extrapolate the number of transformants present in the total transformation mixture from the number of resulting colonies.
2.1.4After growth, collect cells and extract plasmid library pool corresponding to single nucleotide triplet deletion library.Follow procedure described in step 1.4.After this step, the resulting single nucleotide triplet library can be stored as a DNA solution.

Remove Del2 (or Del3
) selection cassette by digesting the cassette insertion library plasmid pool with SchI.This step yields a linearised double or triple nucleotide triplet deletion plasmid library from Del2 or Del3 insertion plasmid library, respectively.Follow procedure outlined at step 2.1.1.

2.2.6
Re-circularise the double or triple nucleotide triplet plasmid library by intramolecular ligation.Follow procedure outlined at step 2.1.2.

2.2.7
Transform puri ed ligation mix to 2.5 to 25 ng of plasmid DNA) into electrocompetent E. coli cells.
Follow procedure outlined at step 2.1.3.

2.2.8
After growth, collect cells and extract plasmid library pool corresponding to double or nucleotide triplet deletion library.Follow procedure described in step 1.4.After this step, the resulting libraries can be stored in the form of DNA solutions.Follow procedure outlined step 3.1.This step yields a linearised plasmid library with cuts randomly distributed within the GOI and allowing the subsequent introduction of nucleotide triplet insertions.

Construction of insertion libraries from TransIns insertion libraries
Separate the resulting fragments by agarose gel electrophoresis and extract the digestion product corresponding to the target vector containing the GOI.Separate the resulting fragments by agarose gel electrophoresis and extract the digestion product corresponding to the target vector containing the GOI.
This step yields a linear plasmid library with random 1, 2 or 3 nucleotide triplet insertions within GOI.
Perform reaction in NEB buffer supplemented with 33 µM of each dNTP (132 µM total dNTPs).Adjust ratio enzyme/DNA accordingly by using 1 unit of enzyme per µg of DNA to be treated.Incubate reaction for 15 min at 25 °C.Stop digestion by adding EDTA (around 10 mM nal) and heating for 20 min at 80 °C.Purify and concentrate DNA product in 10 µl Nuclease-free water.

2.2.6
Re-circularise the insertion plasmid library by intramolecular ligation.
Follow procedure outlined at step 2.2.7 Transform puri ed ligation mix (corresponding 2.5 to 25 of plasmid DNA) into electrocompetent E. coli cells.
Follow procedure outlined at step 2.1.3.

2.2.8
After growth, collect cells and extract plasmid library pool corresponding to single, double or triple nucleotide triplet insertion library.
Follow procedure described in step 1.4.After this step, the resulting libraries can be stored in the form of DNA solutions.
Troubleshooting Note 1: To enable TRIAD, any recognition sequences for MlyI (SchI), NotI and AcuI (Eco57I) in the GOI must be removed either by site-directed mutagenesis (e.g., by introducing a silent mutation that removes an eventual recognition sequences) or by designing and ordering a speci c synthetic gene.Bespoke cloning vectors (i.e., pID-Tet and pID-T7; supplementary les 1 and 2) have previously been assembled to host the GOI during the TRIAD procedure [7] and are available from the authors.
Note 2: In our hands, 1 μl of puri ed and concentrated transposition reaction mix usually yields 10,000 to 50,000 CFUs using 25 μl of commercial electrocompetent cells.The use of high transformation e ciency cells (>10 9 CFUs/μg) is recommended to generate highly diverse libraries at each relevant step of the TRIAD procedure.

Time Taken
TRIAD should span just over 5 days when performing ligations and transformations on the same day (see the timeline in Figure 1).

Anticipated Results
Figures
Target vector: 10 to 50 ng GOI with transposon: calculate quantity depending on fragment size T4 DNA ligase: 5 Weiss units (1 µl) 10X T4 DNA Ligase buffer: 2 µl Nuclease-free water to 20 µl Incubate 30 minutes at room temperature or overnight at 16 °C.Purify and concentrate ligation product in 10 µl nuclease-free water.1.7 Transform puri ed ligation mix (corresponding to 2.5 to 25 ng of plasmid DNA) into electrocompetent E. coli cells.See step 1.3 for transformation and incubation conditions.

2. 1 . 3
Transform puri ed ligation mix (corresponding to 2.5 to 25 ng of plasmid DNA) into electrocompetent E. coli cells.See step 1.3 for transformation conditions.

3. 3
Ligate the InsX selection cassette in the linearised plasmid library obtained at step 3.2.Follow procedure outlined at step 2.2.2.3.4Transform puri ed ligation mix (corresponding to 2.5 to 25 ng of plasmid DNA) into electrocompetent E. coli cells.See step 2.2.3 for transformation and incubation conditions.

3. 5
After growth, collect cells and extract plasmid library pool corresponding InsX insertion library.procedure in step 1.4.3.6 Remove InsX selection cassette by digesting the cassette insertion library plasmid pool with Eco57I.This step yields a linearised double triple nucleotide triplet insertion plasmid library from Ins1, Ins2 or Ins3 insertion plasmid library, respectively.Digestion with Eco57I removes InsX leaving 2-base 3'-overhangs to be removed in the next step.at 37 °C then heat-inactivate for 5 minutes at 80 °C.

Step 2 :
digestion by NotI and MlyI removes TransIns.Step 3: DNA cassettes dubbed Ins1, Ins 3 and Ins3 (with respectively 1, 2 and 3 randomized bp triplets at one of their extremities; indicated in blue) are then inserted between the break in the target gene to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries.Step 4: AcuI digestion and 5'end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target gene.Step 5: Intramolecular ligation results in the reformation of the target gene plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.Purple vertical lines indicate insertions.

Figure 2 Mechanism
Figure 2 This is a of supplementary les associated with this preprint.Click to download.