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. 2023 Feb 20;15(1):2177978. doi: 10.1080/19420862.2023.2177978

Figure 2.

Generation of E. coli SK25 for SpyDisplay using λ red recombination. A shows the use of phage λ Red recombinase to replace the araBAD genes of TG1 E. coli with a SpyCatcher-pIII-containing cassette, followed by excision of a kanamycin resistance gene by FLP recombinase, resulting in E. coli SK25. B shows the expression of SpyCatcher-pIII in SK25 using different concentrations of arabinose for induction by immunoblotting. SpyCatcher-pIII expression increases with increasing arabinose-concentrations, having comparable levels of expression in a range from 0.0005% to 0.05% and showing decreased expression when exceeding concentrations of 0.5%. C shows the production of phages by infecting SK25 with Hyperphage by detecting the presence of SpyCatcher-pIII in these phages by immunoblotting.

Creation of SpyCatcher-pIII expressing E. coli strain SK25.

(a) Phage λ Red recombinase A was used to replace the araBAD genes of TG1 E. coli with a cassette consisting of a kanamycin resistance gene, araC, and SpyCatcher-pIII under control of the pBAD promotor. In the second step, the kanamycin resistance cassette was excised via FLP recombinase. (b) Expression of SpyCatcher-pIII in SK25 bacteria analyzed by immunoblotting. SK25 cells were grown overnight at 22°C in presence of varying concentrations of arabinose and equal numbers of bacteria were lysed. Lysates were probed with anti-M13-pIII followed by sheep anti-mouse IgG (H/L):HRP. For SpyCatcher-pIII, the apparent molecular weight by SDS PAGE of 75 kDa is higher than the calculated molecular weight of 57 kDa. (c) Immunoblot of phages produced by infecting SK25 bacteria with Hyperphage. PEG-precipitated phages were separated electrophoretically, immunoblotted, and detection was performed as described in B.