Site-­‐directed mutagenesis of cell division proteins for contrac5on genera5on in vitro Don Praveen Amarasinghe1, Claire Dow1, David Roper2, Alison Rodger1 1MOAC Doctoral Training Centre & 2School of Life Sciences, The University of Warwick, Coventry, CV4 7AL, U.K. Background Figure 1 The variety of proteins involved in bacterial cell division – reproduced from Vicente et al. 2003 A large number of proteins are known to be involved in bacterial cell division, but very liQle is known about their dynamics throughout the process. The goal of this project was to ins5gate a muta5on in two of these proteins (FtsZ and ZipA), to enable ﬂuorescent tagging and observa5on of their interac5on in the forma5on and contrac5on of the contrac5le Z-­‐ring of a dividing cell. Figure 2 An image of the interac5on between fragments of an FtsZ monomer (red) and Zip A in E. coli – reproduced from PDB (1F47) & Mosyak et al. 2000 Method Figure 3 (right) outlines the mutagenesis protocol. • Four residues in each protein were chosen to be mutated into cysteines. • The QuikChange online primer calculator was used to build the DNA primers required to ins5gate the muta5on. • Dpn1 was used to remove the original methylated DNA from plasmid mixtures aYer PCR. • BL21(DE3) and C43(DE3) cells were used to over-­‐express muta5on-­‐containing FtsZ and ZipA genes. • IPTG was used to induce over-­‐expression via the Lac operon. Figure 3 Summary of the site-­‐directed mutagenesis protocol. Results Three muta5ons were successful according to sequencing results: • FtsZ residue 21 – Glycine to Cysteine. • ZipA residue 328 – Alanine to Cysteine. • A residue coded in the plasmid upstream of ZipA – Phenylalanine to Cysteine. However, although a wide range of DNA polymerases (PfuUltraTM, PhusionTM and Q5TM), annealing temperatures (50 to 72°C) and annealing 5mes (30 to 90 seconds per kb), a very small number of mutagenesis experiments yielded the required muta5on. Next steps • Carry out the mutagenesis with the SPRINP method (Edelheit et al. (2009)), to improve mutagenesis yield. • Protein veriﬁca5on. Ø MALDI to check the amino acid sequence is the one desired. Ø Circular dichroism to check that the secondary structure is s5ll the same as in the wild-­‐type. Ø Linear dichroism to ascertain if the mutated FtsZ will s5ll polymerise. • AQach ﬂuorescent tags to the cysteine residues generated in mutated FtsZ and ZipA and to a cysteine residue already present in another Z-­‐ring protein, ZapA. Figure 4 DNA gel of products of mutagenesis PCR with PhusionTM DNA Polymerase. Lane 1 contains GeneRulerTM. Lanes 2-­‐5, 6-­‐9 and 10-­‐13 correspond to the results for three diﬀerent muta5ons of FtsZ at 4 diﬀerent annealing temperatures. Lane 4 corresponds to muta5on of the residue 21 from glycine to cysteine with annealing temperature 70°C, and indicates the presence of PCR product. This is not apparent in the other lanes. References • • • • Vicente, Miguel and Lowe, Jan. Ring, helix, sphere and cylinder: the basic geometry of prokaryo5c cell division. EMBO Reports, 4(7):655–660, 2003 Mosyak, Lidia; Zhang, Yan; Glasfeld, Elizabeth; Haney, Steve; Stahl, Mark; Seehra, Jasbir and Somers, William S. EMBO Journal, 19(13):3179 – 3191, 2000 Edelheit, Oded; Hanukoglu, Aaron and Hanukoglu. Israel. Simple and eﬃcient site-­‐directed mutagenesis using two single-­‐primer reac5ons in parallel to generate mutants for protein structure-­‐func5on studies. BMC Biotechnology, 9(61): 1–8, June 2009. Knowles, Timothy J.; Finka, Rachael; Smith, Corinne; Lin, Yu-­‐Pin; Daﬀorn, Tim and Overduin, Michael. Membrane Proteins Solubilized Intact in Lipid Containing Nanopar5cles Bounded by Styrene Maleic Acid Copolymer. Journal of the American Chemical Society, 131(22):7484–7485, 2009. • Construct “ar5ﬁcial cells” using the SMALP method outlined in Knowles et al. (2009) containing tagged FtsZ and ZapA in the cytosol and tagged ZipA in the membrane. • Observe the binding of FtsZ polymers to the membrane-­‐bound ZipA (with the aid of ZapA) using confocal microscopy. Acknowledgements The authors would like to thank EPSRC and BBSRC for their con5nued support and funding. Special thanks go to fellow staﬀ and students at MOAC and the structural biology laboratory in the School of Life Sciences.