Supplementary Information for: Engineered synthetic scaffolds for organising proteins within bacterial cytoplasms

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1 Lee et al. Supplementary Information 1 Supplementary Information for: Engineered synthetic scaffolds for organising proteins within bacterial cytoplasms Authors: Matthew J. Lee, 1 Judith Mantell, 2,3 Lorna Hodgson, 2 Dominic Alibhai, 2 Jordan M Fletcher, 4 Ian R. Brown, 1 Stefanie Frank, 5 Wei-Feng Xue, 1 Paul Verkade, 2,3,6 Derek N Woolfson, 2,4,6 * Martin J Warren 1 *. 1 Industrial Biotechnology Centre, School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK 2 School of Biochemistry, University of Bristol, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK. 3 Wolfson Bioimaging Facility, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK. 4 School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, UK. 5 Department of Biochemical Engineering, University College London, Bernard Katz Building, Gordon Street, London WC1E 6BT, UK. 6 BrisSynBio, Life Sciences Building, Tyndall Avenue, Bristol BS8 1TQ, UK. * Correspondence to: Martin J Warren (M.J. Warren@kent.ac.uk) and Derek N Woolfson (D.N.Woolfson@bristol.ac.uk)

2 Lee et al. Supplementary Information 2 Supplementary Fig. 1. Construction of a bacterial cytoscaffold. Hexagonally arrayed, tubular filaments of the PduA* protein (red) carrying half of a de novo designed heterodimeric coiled coil (blue).

3 Lee et al. Supplementary Information 3 Supplementary Fig. 2. TEM analysis of thin sectioned E. coli cells transformed with plyss- PduA*. Scale bar shows 200 nm

4 Lee et al. Supplementary Information 4 Supplementary Fig. 3. TEM analysis of E. coli cells transformed with C-PduA*. Scale bar shows 200 nm.

5 Lee et al. Supplementary Information 5 Supplementary Fig. 4. Western blot analysis of E. coli cells transformed with varying PduA* constructs. Total lysates were analyzed by SDS-PAGE and subsequently western blotted with an anti-pdua* primary antibody in comparison to a molecular weight standard. Cell densities were normalized to an OD 600 of 5 for loading.

6 Lee et al. Supplementary Information 6 Supplementary Fig. 5. Time course of plyss-cc-di-b-pdua* induction with 400 µm IPTG in BL21 * (DE3) cells. (a) pre-induction (b) 1h post induction (c) 2h post induction (d) 3h post induction (e) 4h post induction (f) 6h post induction (g) 20h post induction (h) Control strain transformed with an empty plyss vector. Scale bars show 200 nm

7 Lee et al. Supplementary Information 7 Supplementary Fig. 6. SDS-PAGE of CC-Di-B-PduA* purification. Lane 1 lysate (2 μl), lane 2 supernatant after first centrifugation (2 μl), lane 3 pellet after first centrifugation (5 μl), lane 4 pellet after second centrifugation (5 μl), lane 5 supernatant after second centrifugation (5 μl), lane 6 supernatant after third centrifugation (5 μl), lane 7 pellet after third centrifugation (5 μl), lane 8 supernatant after final centrifugation (5 μl), lane 9 Pellet after final centrifugation (5 μl).

8 Lee et al. Supplementary Information 8 Supplementary Fig. 7. TEM (a), AFM height (b), PeakForce (c) and height profile corresponding to line 1 (panel B) (d) analysis of purified CC-Di-B-PduA* nanotubes.

9 Lee et al. Supplementary Information 9 Supplementary Fig. 8. Confocal analysis of BL21 * (DE3) cells expressing CC-Di-B-PduA* or a control empty vector (plyss) with citrine tagged with CC-Di-A or CC-Di-B or a control C- citrine.

10 Lee et al. Supplementary Information 10 Supplementary Fig. 9. Correlative Light Electron Microscopy of E. coli cells expressing CC- Di-B-PduA* and C-GFP.

11 Lee et al. Supplementary Information 11 Supplementary Fig. 10. (a) Growth curves of strains producing ethanol and control strains shown as OD 600 against time (hours). (b) In vivo ethanol production. Graph shows ethanol content of the growth medium over time. E. coli strain transformed with empty plasmids (pet14b and plyss) ( ), strain producing CC-Di-B-PduA* only ( ), strain producing CC-Di- A-Pdc and CC-Di-A-Adh ( ), strain producing CC-Di-A-Pdc, CC-Di-A-Adh and CC-Di-B- PduA* ( ). Data points represent an average of three independent experiments; standard deviations are represented by error bars.

12 Lee et al. Supplementary Information 12 Supplementary Fig. 11. Western blot analysis of strains producing ethanol and control strain in comparison to a molecular weight marker (M). Total lysates at 24h were adjusted to the same OD 600 (1.6) and analyzed by SDS-PAGE and subsequently western blotted with an anti-polyhistidine primary antibody. (1) E. coli strain transformed with empty plasmids (pet14b and plyss), (2) strain producing CC-Di-B-PduA* only, (3) strain producing CC-Di- A-Pdc and CC-Di-A-Adh, (4) strain producing CC-Di-A-Pdc, CC-Di-A-Adh and CC-Di-B- PduA*.

13 Lee et al. Supplementary Information 13 Supplementary Fig. 12. TEM analysis of ethanol producing strains after 24 hours. (a) E. coli strain transformed with empty plasmids (pet14b and plyss), (b) strain producing CC- Di-B-PduA* only, (c) strain producing CC-Di-A-Pdc and CC-Di-A-Adh, (d) strain producing CC-Di-A-Pdc, CC-Di-A-Adh and CC-Di-B-PduA*.

14 Lee et al. Supplementary Information 14 Supplementary Fig. 13. Confocal image of strain expressing C citrine MinD; scale bar is 5 µm.

15 Lee et al. Supplementary Information 15 Supplementary Fig. 14. TEM micrographs of strains producing CC-Di-B PduA* only (a); CC-Di-B PduA* plus the C citrine MinD control (b), arrows indicate transverse filaments. Zoom in of areas 1 and 2 in panels a and b respectively (c and d).

16 Lee et al. Supplementary Information 16 Supplementary Tables Supplementary Table 1. Strains used in this study Strain Genotype Source JM109 enda1, reca1, gyra96, thi, hsdr17 (rk, mk+), rela1, supe44, Δ(lac-proAB), [F, trad36, proab, laqiqzδm15] Promega BL21 * (DE3) F ompt hsdsb (rb mb ) gal dcm (DE3) Novagen Supplementary Table 2. DNA synthesized for this study Name CC-Di-A CC-Di-B Control DNA / translated sequence (coiled-coil peptides underlined) TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATATGGGTAGCG AAATTGCCGCGCTGGAAAAAGAAAACGCTGCCCTGGAGCAGGAAAT CGCGGCACTGGAGCAGGGTAGCGGCAGCGGTAGCACCATGGGCAG CAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGC AGCCATATG MGSEIAALEKENAALEQEIAALEQGSGSGSTMGSSHHHHHHSSGLVPR GSHM TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATATGGGTAGCA AAATCGCCGCACTGAAAAAGAAAAACGCTGCGCTGAAACAGAAAATT GCCGCGCTGAAACAGGGTAGCGGCAGCGGTAGCACCATGGGCAGC AGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCA GCCATATG MGSKIAALKKKNAALKQKIAALKQGSGSGSTMGSSHHHHHHSSGLVPR GSHM TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATATGGGTAGCG GTAGCGGCAGCGGTAGCACCATGGGCAGCAGCCATCATCATCATCA TCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATG MGSGSGSGSTMGSSHHHHHHSSGLVPRGSHM MinD ACTAGTGGTTCGGGATCGCCGTTGCAAGTCTTGGAGGAACAAAACA AAGGGATGATGGCTAAAATCAAATCATTTTTCGGTGTACGTTCCTAA CCTAGGTTTGGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAG TTGGCTGCTGCCACCGCTGAGC TSGSGSPLQVLEEQNKGMMAKIKSFFGVRS

17 Lee et al. Supplementary Information 17 Supplementary Table 3. Plasmids Used in this study Plasmid name Description Source pet14b Overexpression vector containing N-terminal hexahistidine-tag, modified to include an SpeI site 5 of BamHI Novagen plyss Basal expression suppressor Novagen plyss_2 pet3a_pdua* plyss_pdua* pet_cc_di_a pet_cc_di_b pet_c pet_cc_di_a_pdua* pet_cc_di_b_pdua* pet_c_pdua* pet-tbad-coba pet3a-mcherry- PduABB JKNU pet-tbad-mcherry- PduABJKNU. plyss-mcherry- PduABB plyss-prha-mcherry- PduAB plyss_tbad_mcherry _PduABJKNU plyss_cc_di_a_pdu A* Basal expression suppressor containing the T7 promoter-mcs-t7 terminator cassette from pet14b PCR product of pdua* ligated into the NdeI and SpeI sites of pet3a (4 bp removed from 3' end of pdua, this allowed non-coding DNA from the vector to be translated) NdeI/SpeI fragment of pet3a_pdua* ligated into NdeI/SpeI sites of plyss_2 Synthesised CC_Di_A gene fragment cloned into XbaI/NdeI sites of pet14b Synthesised CC_Di_B gene fragment cloned into XbaI/NdeI sites of pet14b Synthesised control (C) gene fragment cloned into XbaI/NdeI sites of pet14b NdeI/SpeI fragment of plyss_pdua* cloned into NdeI/SpeI sites of pet_cc_di_a NdeI/SpeI fragment of plyss_pdua* cloned into NdeI/SpeI sites of pet_cc_di_b NdeI/SpeI fragment of plyss_pdua* cloned into NdeI/SpeI sites of pet_c XbaI/HindIII fragment of pet3a-mcherry- PduABJKNU cloned into XbaI/HindIII sites of pet-tbad-coba Exponential Megapriming PCR (EMP) and splicing by Overlap Extension (SOE) to insert BglII site and prha promoter at former T7 promoter site of plyss-mcherry-pduabb BglII/SpeI fragment of pet-tbad-mcherry- PduABJKNU cloned into BglII/SpeI sites of plyss-prha-mcherry-pduab BglII/SpeI fragment of pet_cc_di_a_pdua* cloned into BglII/SpeI sites of Parsons et al., 2008 Parsons et al., 2010 Gift from E. Deery Parsons et al., 2010 Parsons et al., 2010

18 Lee et al. Supplementary Information 18 plyss_cc_di_b_pdu A* plyss_c_pdua* pet_cc_di_a_citrine pet_cc_di_b_citrine pet_c_citrine pet_cc_di_a_mcherr y pet_cc_di_b_mcherr y pet_c_mcherry pet_cc_di_a_citrine _CC_Di_A_mCherry pet_c_citrine_c_mc herry pet23b-pdc pet23b-adh pet_cc_di_a_pdc pet_cc_di_a_adh pet_cc_di_a_adh_c C_Di_A_Pdc plyss_tbad_mcherry_pduabjknu BglII/SpeI fragment of pet_cc_di_b_pdua* cloned into BglII/SpeI sites of plyss_tbad_mcherry_pduabjknu BglII/SpeI fragment of pet_c_pdua* cloned into BglII/SpeI sites of plyss_tbad_mcherry_pduabjknu Citrine PCR product ligated into NdeI/SpeI sites of pet_cc_di_a Citrine PCR product ligated into NdeI/SpeI sites of pet_cc_di_b Citrine PCR product ligated into NdeI/SpeI sites of pet_c mcherry PCR product ligated into NdeI/SpeI sites of pet_cc_di_a mcherry PCR product ligated into NdeI/SpeI sites of pet_cc_di_b mcherry PCR product ligated into NdeI/SpeI sites of pet_c XbaI/EcoRI fragment from pet_cc_di_a_mcherry ligated into SpeI/EcoRI sites of pet_cc_di_a_citrine XbaI/EcoRI fragment from pet_c_mcherry ligated into SpeI/EcoRI sites of pet_c_citrine NdeI/EcoRI fragment of pet23b-pdc cloned into NdeI/EcoRI sites of pet_cc_di_a NdeI/EcoRI fragment of pet23b-adh cloned into NdeI/EcoRI sites of pet_cc_di_a XbaI/EcoRI fragment from pet_cc_di_a_pdc ligated into SpeI/EcoRI sites of pet_cc_di_a_adh Lawrence et al., 2014 Lawrence et al., 2014

19 Lee et al. Supplementary Information 19 Supplementary Table 4. Oligonucleotides used in this study, restriction sites are underlined Name Sequence 5 3 Citrine_NdeI_FW CACCATATGGTGAGCAAGGGCGAGGAGC Citrine_SpeI_RV CACACTAGTTTACTTGTACAGCTCGTCC mcherry_ndei_fw CAGCATATGGTGAGCAAGGGCGAGG mcherry_spei_rv GACACTAGTTTACTTGTACAGCTCGTCCATGC Citrine_SpeI_NoStop_RV CACACTAGTCTTGTACAGCTCGTCC

20 Lee et al. Supplementary Information 20 Supplementary Movies Supplementary Video 1. Tomography of CC-Di-B-PduA filaments and automated microtubule tracing. Supplementary Video 2. Refined tracing model on a small area of the tomogram shown in movie S1.