ALUMBRADO PÚBLICO E ILUMINACIÓN EXTERIOR
535.4.5 CÁLCULO DE LAS CARACTERÍSTICAS DE CALIDAD DE LA LUMINANCIA
RNase A degradation of RNCs. FLRNC was produced and purified according to
Material and Methods section 2.2. After use of the Superdex75 column to separate FLRNC from FLP, the FLRNC-containing fractions were concentrated and incubated with RNAse A (50 μg/ml) and 25 mM EDTA for 1 hour at 37 °C. The resulting sample was spun down for 10 minutes at 18000 RCF and the supernatant was subsequently run on a Superdex75 column while fractions were collected during the run. The frac- tions were characterized using a 15% SDS-PAGE gel and subsequently blotted with anti-flavodoxin.
Elution of ribosomes versus released nascent chains. To verify the elution patterns of ribosomes versus FLP, first empty ribosomes (purchased from New En- gland Biolabs (NEB)) were run on a Superdex75 column. Subsequently FLP (purified in the experiment described above) was mixed with the empty ribosomes and this mixture was run over the Superdex75 column. Lastly purified FLP was run over the same Superdex75 column.
Expression of terminally truncated variants of flavodoxin without fusion to the Smt3-domain and the stalling sequence. The pUC18 plasmid containing the flavodoxin gene was used to construct two protein variants, 1-174 and 1-169, in which the stop codon has been moved N-terminally. These protein variants mimic the -5P (1-174) and -10P (1-169) molecules, respectively, but lack the StrepII-tag, Smt3-domain and SecM sequence. Both protein variants (named -5 and -10, respec- tively), along with full-length flavodoxin (FL) and empty vector pUC18 (pUC) were expressed in E. coli (strain TG2) grown on TB or minimal medium. Half of the cor- responding cultures were induced with IPTG for a period of 16 hours. Gel samples were taken before and after induction. The cells in these samples were lysed and separated into supernatant and pellet fractions by centrifugation. Supernatant and pellet samples were analysed by running them on a 15% SDS-PAGE gel and subse- quent blotting with anti-flavodoxin.
Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome
Relative absorbance at 260 nm & 280 nm
Relative elution volume
Relative absorbance at 450 nm 1.0 0.8 0.6 0.4 0.2 0.0 30x10-3 25 20 15 10 0.2 0.4 0.6 0.8 1.0 0 5 0.0 A A450 A280 A260 FLP 0.05 0.04 0.03 0.02 0.01 0.00 0.7 0.6 0.5 0.4 0.3 5x10-3 4 3 2 1 0 FLP 37 50 B C Relative absorbance at 450 nm
Relative absorbance at 260 nm & 280 nm
Figure S1 Disruption of FLRNC by RNase A results in elution of nascent FL at relative volume of FLP. Shown are Superdex75 elution profiles, with all absorbance traces normalized relative to the maximum absorbance at 260 nm. Absorbance at 450 nm tracks FMN incorporation. Elution volumes are normalized to the total column vol- ume Vt. A, FLRNC incubated for 1 hour with RNase A. Due to degradation of rRNA, the RNC peak has vanished, resulting in several smaller peaks corresponding to ribo- somal proteins and rRNA. B, Expansion of (A) reveals the presence of a peak with ab- sorbance at 450 nm, which elutes at the same relative volume as FLP. Dashed vertical lines designate collected fractions during the run. C, Collected fractions are analysed by Western blot probed with anti-flavodoxin. Fractions corresponding to the peak with absorbance at 450 nm indeed contain FLP.
82 Chapter 3 1.0 0.8 0.6 0.4 0.2 0.0 30x10-3 25 20 15 10 5 0 1.0 0.8 0.6 0.4 0.2 0.0
Relative absorbance at 260 nm & 280 nm
30x10-3 25 20 15 10 5 0 Relative absorbance at 450 nm 1.0 0.8 0.6 0.4 0.2 0.0 0.6 0.5 0.4 0.3
Relative elution volume
30x10-3 25 20 15 10 5 0 C A B A 260 A450 A280
Figure S2 FLP does not co-elute with intact, empty ribosomes. Shown are Super-
dex75 elution profiles, with all absorbance traces normalized relative to the maxi- mum absorbance at 260 nm of empty ribosomes. Absorbance at 450 nm tracks FMN incorporation. Elution volumes are normalized to the total column volume Vt. A, Empty ribosomes (purchased from NEB). B, Mixture of empty ribosomes and puri- fied FLP. C, Purified FLP.
Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome
FL
pUC -10 -5
Sup Pel Sup Pel MM TB 0 ON 0 0 0 i ON i ON i ON i ON u ON u ON u ON u
Figure S3 Expression of terminally truncated variants of flavodoxin. Shown are Western blots probed with anti-flavodoxin. Three variants of flavodoxin were pro- duced. The first one is full-length protein (FL). In the other two variants the regular stop codon has been moved N-terminally to create protein variants 1-174 (-5) and 1-169 (-10). As a control, the empty vector containing no flavodoxin gene (pUC) was also tested. All four constructs were transformed into E. coli (strain TG2) and grown either in nutrient-rich medium (TB) or minimal medium (MM). Samples were taken before induction (0), after induction for 16 hours (ONi) or after growth for 16 hours without induction (ONu). Subsequently, cells of the samples were lysed and both supernatant (Sup) and pellet (Pel) fractions were analysed on Western blot. Due to a leaky promoter in the vector, protein is produced in un-induced samples. For full-length flavodoxin, protein is found in supernatant and pellet fractions of cells grown on both TB and MM medium. -5 and -10 are detected only in the pellet of cells grown on TB medium. Due to lower expression of the -5 and -10 protein in cells grown on MM medium compared to on TB medium, the protease machinery is able to degrade all produced protein in the first situation, but not in the second one. As expected, the empty vector does not show production of flavodoxin, though there is some cross-reactivity of the flavodoxin antibody with E. coli proteins in the fractions.
FL TB FL MM -10 TB -10 MM -5 TB pUC TB
Figure S4 FMN content of overexpressed flavodoxin is reflected in the colouring of pelleted cells. Three variants of flavodoxin (FL, -5 and -10) and a vector containing no flavodoxin gene (pUC) were transformed in E. coli (strain TG2) and grown and induced for 16 hours. Subsequently, cells were pelleted and checked for their colour. Expression of FL in TB results in a blue-greenish colour due to the semiquinone form of FMN. TB and MM denote the medium in which the cells were induced, i.e., nutrient-rich (TB) or minimal medium (MM). FL and -5 induced in TB show a darker blue-greenish coloured cell pellet than -10 (which is incapable of binding FMN) or pUC (which does not express flavodoxin), indicating binding of FMN. FL has a darker coloured cell pellet than -5, signifying higher FMN content. Due to lower growth in MM medium, the cell pellet in these samples is not large enough for the colour change between FL and -10 to be visible.