The location for this initial genomic landing pad was selected as the non- essential E. coli pepA gene, due to it having been previously mutated without negative impact to the cell (Reijns et al., 2005) and the availability of a pepA knockout assay. The pepA gene encodes an aminopeptidase, which has a role as an accessory protein in the XerC/XerD recombinase dimer resolution mechanism (Reijns et al., 2005).
The φC31 integrase landing pad was inserted into the pepA locus, replacing the entire gene, via the λ Red recombineering method. This is a method of facilitating integration of a PCR product into the genome via homologous recombination. The desired sequence to be integrated is amplified by PCR with flanking regions of homology to the targeted gene. The PCR product is then introduced into E. coli cells expressing the λ Red proteins, which block activity of cellular endonucleases and promote homologous recombination. A diagram illustrating how this mechanism acts to replace the pepA gene with a homology-added φC31 integrase LP (EmR) PCR product is shown in Figure 3.3.
Figur
e 3.3
: Using the
λ Red r
ecombineering method, a PCR pr
oduct consisting of the
φ
C31
integrase LP (
EmR
) flanked by homology sequences to the start and end of the
pepA
gene is
integrated into the
E. coli
genome, r
esulting in the complete r
eplacement of
pepA
with the
landing pad cassette, illustrated her
e in its genomic location between genes
yjgP
and
holC
E. coli DS941 cells were transformed with λ Red recombineering plasmid pKOBEG-A (Chaveroche et al., 2000), expressing λ Red proteins (Gam, a nuclease inhibitor; Exo, a 5’ to 3’ exonuclease; and Beta, which protects and promotes annealing to the ssDNA created by Exo) under control of the pBAD promoter which is induced by the addition of arabinose. Successful
transformants were isolated by selecting for ampicillin resistance carried by the pKOBEG-A vector. This plasmid is designed to be temperature-sensitive in order to allow removal following successful recombineering; this occurs due to the presence of a mutation in the pSC101 origin backbone which prevents replication of the plasmid at temperatures above 37°C (Hashimoto-Gotohi & Sekiguchi, 1977). Therefore all growth steps consequent to transformation were conducted at 30°C in order to maintain the pKOBEG-A plasmid in the cells. These DS941 cells containing pKOBEG-A were then ready for induction of Red functions and transformation with the LP recombineering PCR product.
Primers were designed to amplify the EmR gene with φC31 integrase attBTT
and attBTC sites flanking up- and downstream respectively, along with 50 bp
homology sequences to the start and end of the pepA gene. These primer sequences are shown in Methods Section 2.4. Using these primers, the recombineering cassette (LPC31) was amplified using a Phusion polymerase protocol nicknamed ‘Phussean' (Methods Section 2.13.), which has been optimised for performance with larger primers such as the oligonucleotide primers synthesised for this PCR (123 bp forward primer, 129 bp reverse primer; see Methods Section 2.4). The PCR template used was the EmR gene found on the genome of the E. coli DS941-derived strain π1 (see Methods Section 2.5), a strain previously engineered by this lab, in a method that allows amplification directly from the genome by resuspending a colony in water and adding it to the reaction set up (Methods Section 2.13.)
Purification of the PCR product was done in one of two ways during recombineering experiments.
At first, PCR reactions were directly run in their entirety on a 1% agarose gel and the correctly sized DNA band (1253 bp) was excised and extracted using a QIAgen Gel Extraction kit. However, after several unsuccessful attempts at recombineering with this PCR product sample, it was thought that exposure to UV during excision of the agarose gel band was having a damaging effect on the DNA, thus decreasing efficiency of the homologous recombineering process. Subsequent PCR products of the LP recombineering fragments were purified using a QIAgen PCR Purification kit immediately after completion of the PCR, and a sample of this eluted DNA was run on a 1% agarose gel to confirm the presence and size of the DNA. This avoided damage to the DNA by UV radiation and increased chances of successful recombineering in
subsequent protocols.
The φC31 LP recombineering PCR product was introduced by electroporation into DS941/pKOBEG-A cells that had been induced by L-arabinose for λ Red functions. Following transformation and recovery at 30°C, successful EmR LP recombinants were selected by their resistance to erythromycin. Initially the efficiency of recombineering was seen to be very low, with many repetitions of the protocol resulting in no observed EmR-positive colonies on
erythromycin LB agar plates.
After many iterations of this method, when successful erythromycin-resistant colonies were finally seen (healthy colonies on LB + erythromycin plates following electrotransformation) colony counts were very low, with a
maximum of 4 colonies per plate observed. It should be noted that of these successful plates, it was found that incubating agar plates at 37°C rather than 30°C resulted in an improved rate of recombineering. This had previously been suggested to be an improved temperature for the activity of the λ Red proteins, and while the higher temperature decreases the ability of pKOBEG-A to proliferate, sufficient expression of the λ Red proteins should be
In order to clear transformed cells of the pKOBEG-A temperature-sensitive plasmid, erythromycin-resistant colonies were streaked onto LB agar plates plus erythromycin and grown at 42°C. At this temperature, replication of the pKOBEG-A plasmid is inhibited and the vector does not proliferate. After several generations the temperature-sensitive plasmids should therefore eventually be lost from the cells. After two rounds of re-streaking and growth at 42°C, cells were tested for loss of the pKOBEG-A plasmid by streaking onto LB agar plus ampicillin. Absence of growth on these plates indicated the successful loss of the plasmid.