Expression and cloned into pet28b with nde i and hind

Info icon This preview shows pages 10–11. Sign up to view the full content.

expression and cloned into pET28b with Nde I and Hind III restriction sites. The plasmid containing MPH was transformed into E. coli BL21 (DE3) and the resulting colonies were grown overnight at 37 u C in LB media supplemented with 50 m g/ml kanamycin. For overexpression, 1 L LB cultures supplemented with 50 m g/ml kanamycin were subcultured from the overnight culture at 1% (v/v) and grown at 37 u C to an OD 600 of 0.5–0.7. The cells were chilled on ice for 15 minutes. Protein expression was induced with 1 mM isopropyl- b -D-thiogalactopyr- anoside at 16 u C for 16 hours. The cells were harvested and stored at 2 20 u C until further use. For protein purification, the cells were resuspended in lysis buffer (50 mM HEPES, 300 mM NaCl, 10 mM Imidazole, pH 7.5), 1 mM phenylmethanesulfonylfluoride and 1 m g/ml pancreatic-bovine DNase and cells were lysed using T-S series cell disrupter. Protein was purified by standard immobilized metal affinity chromatography followed by gel filtration and stored in 10% glycerol at 2 20 u C. Steady State Kinetic Analysis of MPH(2 9 ) Kinetic parameters of MPHs were determined in triplicate using Pyruvate Kinase/Lactate Dehydrogenase coupled assay [53]. Phosphorylation of macrolides by MPHs was monitored by absorbance of NADH (340 nm) in 96 well format using a SpectraMax reader. The reaction was initiated with macrolide or nucleotide (GTP). When monitoring macrolide dependence, 200 m M GTP was used and the macrolide concentration ranged from 3.2 m M and 400 m M. For nucleotide dependence, 200 m M macrolide was used and the final concentrations of GTP ranged from 7.82 m M to 2000 m M. All reactions were performed in triplicate. The initial rates were fit to equation 1 or 2 (substrate inhibition) using Grafit 4.0 software (Erithacus Software, Staines, UK): v ~ V max : ½ S = K m z ½ S ð 1 Þ v ~ V max ± S = ( K m z S ± (1 z S = K i )) ð 2 Þ Growth Analysis of Paenibacillus lautus LC231 with Daptomycin Analyses were performed in 96-well flat bottom plates (200 m l volume total) using 50% TSB supplemented with 1.25 mM CaCl 2 as a growth medium. Inocula represented 1:200 dilutions of an overnight culture, standardized to an OD 600 of 0.1. The following conditions were tested in duplicate: ( 1 ) no daptomycin, ( 2 ) 4 m g/ml daptomycin and ( 3 ) 4 m g/ml daptomycin added after 10.5 hours (early log phase). Plates were incubated while shaking at 30 u C and the data was collected as an OD 600 every 30 minutes using a Tecan Sunrise plate reader. Similar studies were conducted with a surface strain of P. lautus (ATCC 43898) except that the organism was grown in full strength TSB. Supporting Information Text S1 Supporting materials and methods. (DOCX) Figure S1 Lechuguilla cave microbiome. A) Chart of the distribution of bacterial isolates sampled in this work. B) Consensus phylogram of bacterial strains used in this study. The tree was created using 16 S rRNA gene sequences. Node values represent the likelihood of the represented partition at each branch (based on 1000 maximum likelihood bootstrap analyses).
Image of page 10

Info icon This preview has intentionally blurred sections. Sign up to view the full version.

Image of page 11
This is the end of the preview. Sign up to access the rest of the document.

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern