1 Chapter 1: Introduction
2.7 Structural determination of CysB by Small Angle X-ray Scattering (SAXS)
2.7.1 SAXS Sample preparation
Samples were prepared for SEC-SAXS analysis by purification methods detailed in Section 2.4. The third and fourth sets of samples had an altered purification method which saw the addition of 10 % v/v glycerol in all purification buffers. A high salt wash step during IMAC was also included with 4 M NaCl in lysis buffer (50 mM Tris pH 8.0, 4 m NaCl, 20 mM
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imidazole, 10 % v/v glycerol) alongside two incubations with Benzonase® Nuclease (Sigma- Aldrich), to the resuspended pellet and pooled IMAC fractions, to attempt to remove any bound nucleic acids from the sample. Where samples were sent for analysis, final volume of 200 μL of each concentrated sample was sent on ice with corresponding SEC buffers to the Australian SAXS/WAXS BeamLine (Ryan et al., 2018). Upon arrival at the Australian SAXS/WAXS BeamLine, samples were stored at 4°C until analysis. Samples were centrifuged and filtered to remove any aggregation that may have occurred during transport and storage of samples.
Where samples were analysed in person, ample CysB sample was concentrated to 2 mg.mL-1 and transported on ice to the Australian Synchrotron. Upon arrival samples were stored at room temperature, protected from light until analysis. Further concentration (5 000 g, 8°C), centrifugation and filtering ensured CysB was free of visible aggregation with Denovix®
measurements giving precise information about the concentration of CysB loaded onto the column.
2.7.2 SAXS Data Collection
Measurements were performed at the Australian Synchrotron SAXS/WAXS Beamline (Ryan et al., 2018) equipped with a Dectris-Pilatus3-2M detector. Samples were run via SEC-SAXS analysis, where 50 μL of sample was loaded onto a Superdex 200 Increase 5/150 GL (Cytiva) column at a flowrate of 0.4 mL.min-1 with co-flow in place (Kirby et al., 2016). The column was pre-equilibrated with the corresponding buffer, SEC buffer (50 mM Tris pH 8.0, 200 mM NaCl, 10% v/v glycerol) or SEC buffer with 10 mM final concentration N-acetylserine inducer.
Measurements were taken with a sample detector distance of 2.00 m at 12°C with an X-ray wavelength of 1.0322 Å. Data was collected as samples flowed through a 1.5 mm thin-walled glass capillary at one second intervals with an inline UV trace recorded.
2.7.3 SAXS Data Analysis
Several programs from the ATSAS Suite (Manalastas-Cantos et al., 2021) were used in conjunction with the Australian Synchrotron Scatterbrain software to analyse the SEC-SAXS data. UV traces from each sample run were inspected in GraphPad Prism version 9.4.1 for windows to confirm uniform species and give an initial indication on data quality. Buffer subtraction to deconvolute peaks was performed in CHROMIXS from the ATSAS Suite (Panjkovich & Svergun, 2018) with manual inspection of image files undertaken in Scatterbrain to inform data quality. Primus from the ATSAS Suite was used to assess data quality by visual analysis of the Guinier plot, Kratky plot and Porod distribution, while plots
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were made using GraphPad Prism version 9.4.1 for windows. Manipulation of the Porod distribution by an artificially large Rmax allowed any subtle aggregation to be detected in samples.
2.7.4 SAXS Model Building
The Porod distribution with an appropriate Rmax was used to generate an output file for each sample for theoretical modelling in DAMMIN (Svergun, 1999), DAMMIF (Franke & Svergun, 2009) and GASBOR (Svergun, Petoukhov, & Koch, 2001) from the ATSAS Suite. Averaging of DAMMIF models and generation of a surface model from scattering data was performed using DAMAVER (Volkov & Svergun, 2003). A theoretical scattering pattern was obtained from CRYSOL from the ATSAS Suite (Franke et al., 2017) using the final X-ray crystallography model.
2.8 cysB genomic deletion in Neisseria gonorrhoeae
Genomic deletion of cysB was attempted to probe the hypothesised essentiality of the cysB gene in N. gonorrhoeae.
2.8.1 Construct for cysB genomic deletion in N. gonorrhoeae
The DNA template for homologous recombination was designed by Dr Joanna Hicks using Geneious Prime (Biomatters) to contain 150 bp of flanking sequence (corresponding to 150 bp upstream and downstream of the cysB gene respectively) either side of a kanamycin resistance gene (kanR). The DNA construct was ordered as a Geneblock from Twist Bioscience (USA).
Lyophilised DNA was resuspended in TE buffer to a final concentration of 20 ng.μL-1 for use as a DNA template in subsequent PCR reactions. Primers were designed using Geneious Prime (Biomatters) and ordered from IDT (USA) to amplify the entire DNA construct (CysB KO
FWD: 5’- ATAGTCTATCATGCCGAAA -3’ and CysB KO REV: 5’-
CCTGTACGAACATTTCAGAC -3’) which can be found in Appendix AB.5. The construct was purified using a QIAquick® PCR purification kit (Qiagen) following manufacturer’s instructions.
2.8.2 Spot transformations of N. gonorrhoeae
Purified PCR DNA products (prepared as per Section 2.5.6) were pooled and combined with ultra-distilled water to a final concentration of 20 ng.μL-1. Two 10 μL amounts of PCR product were spotted onto GCB agar (Appendix AB.5) and left to soak into the agar and dry. This was repeated onto New York agar (Fort Richards, New Zealand). A viable N. gonorrhoeae colony
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was taken from fresh GCB agar and streaked across the plates through each spot of DNA and incubated at 37°C for 48 hours. Single colonies that grew within the spotted DNA circles from both GCB and New York agar were streaked onto GCB agar supplemented with 50 μg.mL-1 kanamycin and incubated at 37°C for 48 hours to select for integration of the DNA construct into the N. gonorrhoeae chromosome. Primers were designed by Dr Joanna Hicks to confirm integration of the cysB knockout construct via colony PCR of any positive transformants (primers can be found in Appendix AB.5).
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