Site-specific chromosomal integration of large synthetic constructs

Abstract

We have developed an effective, easy-to-use two-step system for the site-directed insertion of large genetic constructs into arbitrary positions in the Escherichia coli chromosome. The system uses lambda-Red mediated recombineering accompanied by the introduction of double-strand DNA breaks in the chromosome and a donor plasmid bearing the desired insertion fragment. Our method, in contrast to existing recombineering or phage-derived insertion methods, allows for the insertion of very large fragments into any desired location and in any orientation. We demonstrate this method by inserting a 7-kb fragment consisting of a venus-tagged lac repressor gene along with a target lacZ reporter into six unique sites distributed symmetrically about the chromosome. We also demonstrate the universality and repeatability of the method by separately inserting the lac repressor gene and the lacZ target into the chromosome at separate locations around the chromosome via repeated application of the protocol.

Figure 1.

Representative recombineering efficiency as a function of insert size. 1000–4500-bp inserts containing the neo gene and bearing 50-bp flanking homology regions were inserted into the lacZ gene of strain K-12 MG1655 pTKRED via the method of Datsenko and Wanner (6). Cells were plated on LB agar + 25 μg/ml kanamycin, and the number of successful recombinants quantified as the number of resulting white colonies.

Figure 2.

(a) Plasmids used in the integration protocol. The sequence size given is for pTKIP-neo; neo is exactly replaced with various antibiotic resistance genes for alternate versions of pTKIP. Small green boxes are I-SceI restriction sites; landing pad regions 1 and 2 are small red boxes labeled LP1 and LP2 respectively. (b) Annotated sequence of the pTKIP MCS showing LP1, available restriction sites, and the first four bases of the adjacent FRT site. (c) Strategy for large construct chromosomal integration. Step 1: the host strain is transformed with the helper plasmid pTKRED, bearing I-SceI endonuclease (green) and λ-Red (red). Linear landing pad fragments (yellow) are integrated into the chromosome at the desired location (black squares) when λ-Red expression is induced by IPTG. Step 2: the host strain is transformed with pTKIP bearing the fragment (purple) to be inserted into the landing pad. I-SceI expression is induced via the addition of l-arabinose, and the I-SceI recognition sites (green) in the donor plasmid and chromosome are cleaved. Integration of the fragment is facilitated by IPTG-induced λ-Red expression. Step 3: pTKRED is cured by growth at 42°C and screening against spectinomycin resistance.

Figure 3.

pTKIP is cured by in vivo I-SceI cleavage. Circles indicate the number of cells retaining pTKRED, squares indicate retention of pTKIP-neo. Normalized cfu is the ratio of surviving colonies on plates containing the appropriate antibiotic (100 μg/ml spectinomycin and 25 μg/ml kanamycin for pTKRED and pTKIP, respectively) to surviving colonies on LB plates without selection.

Figure 4.

(a) Chromosomal insertion positions. Fragments were inserted into six positions distributed symmetrically about the E. coli chromosomal origin of replication oriC. Insertion positions are between the marked genes at each position (dots). (b) Verification of chromosomal insertions. Colony PCR across insertion junctions for insertion of the P_lacI-lacI:venus:T1-neo cassette (IvT-neo; 2.1-kb band) into the nth-ydgR position and the P_LlacO1-lacZ-cat cassette (O1Z-cat; 5-kb band) at each site. The insertion of both the IvT-neo and O1Z-cat cassettes into the nth-ydgR position was accomplished by a single 7-kb insertion bearing the neo marker. Lanes 2–8 are MG1655 Δlac negative controls in the following order: lane 2: nth IvT-neo; lanes 3–8: O1Z in alphabetical order of insertion positions, corresponding to positive insertion lanes 10–20.

Figure 5.

(a) Verification of large chromosomal insertions. Colony PCR of 16 randomly picked colonies obtained from insertion of IvT-O1Z-neo between atpI and gidB. Lane 2: MG1655 Δlac pTKRED negative control atpI proximal junction. Lane 3: MG1655 Δlac pTKRED negative control gidB proximal junction. Lanes 5–20 and 22–37 are IvT-O1Z-neo cassette integrants. Lanes are alternating pairs of atpI proximal junctions (2.1-kb bands; lacI:venus:T1 amplified) and gidB proximal junctions (5-kb bands; lacZ-neo amplified) for each clone. (b, c) Alignment of insertion junction sequences obtained from first eight clones shown in (a). (b) Junction proximal to atpI. (c) Junction proximal to gidB. Sequences of the flanking chromosomal regions, 25-bp landing pad regions, and the inserted fragment are labeled and indicated by a black, red or purple underscore, respectively. Mismatches to the expected sequence are highlighted in blue.

Figure 6.

I-SceI-induced chromosomal breaks are not lethal in the presence of λ-Red. Solid lines are linear regression fits used to calculate doubling time. Circles: MG1655 Δlac pTKRED without landing pad, doubling time 43 min; triangles: essQ-cspB landing pad insertion, doubling time 1.5 h; squares: atpI-gidB landing pad insertion, doubling time 8.3 h.