Additional file 1 of RuBisCO activity assays: a simplified biochemical redox approach for in vitro quantification and an RNA sensor approach for in vivo monitoring
Creators: name: Faisal, Muhammad
name: Sarnaik, Aditya P.
name: Kannoju, Nandini
name: Hajinajaf, Nima
name: Asad, Muhammad Javaid
name: Davis, Ryan W.
name: Varman, Arul M.
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Molecular Biology
subject: Biotechnology
subject: Environmental Sciences not elsewhere classified
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Statistics
subject: FOS: Mathematics

description: Additional file 1: Figure S1. In vitro biochemical assay: Preliminary experiments were performed to estimate the effectiveness of using the cell’s native enzymes for performing the NADH-based spectrophotometric RuBisCO activity assay. The assay was performed by externally supplementing the reaction with two crucial substrates; RuBP and NADH. Simultaneously, two reaction controls were introduced; a without RuBP, with NADH, and b without NADH, with RuBP. The results showed in the plot indicated that there was negligible or no increase in the mM of NAD+ (product of NADH oxidation) for the two controls, where the test sample containing both the substrates showed prominent increase in NAD+ concentration, confirming the in vitro functionality of the assay. The graph for the control reaction without RuBP, further indicated that contribution from the other metabolic pathways to the NADH oxidation was relatively low and below the detection limit of the assay. Figure S2. Standard curve for NADH concentration vs Absorbance at 340 nm. Figure S3. Analysis of the functioning of the ADP-specific RNA sensor. A Functioning of the ADP-specific RNA sensor was confirmed by directly adding 2 mM ADP to the reaction mixture containing the HEPES buffer (pH 7.4) and in vitro transcribed RNA sensor, followed by monitoring its fluorescence. Increase in the fluorescence (subtracting 0 min fluorescence) confirmed the positive response of the sensor to ADP. B Background fluorescence or ADP contribution from other metabolic reactions was analyzed using the control assays. The assay mixture contained 0.5 mM RuBP as the first RuBisCO substrate, 2 mM ATP as the cofactor, and cell lysate as the source of pathway enzymes, where the plate was incubated in a CO2 incubator with 5% CO2 (second inorganic C substrate for RuBisCO). To prove the effectiveness of the assay, three different controls were introduced a) with ATP and CO2, but without RuBP, b) with RuBP and CO2, but without ATP, and c) with ATP and RuBP, but without incubating in 5% CO2. The results showed in the plot indicated that there was no increase in the fluorescence for the three controls. No increase in the fluorescence for the control reactions without RuBP, and without 5% CO2 incubation, further indicated that ADP contribution from the other metabolic pathways to the fluorescence due to ATP hydrolysis was below the detection limit of the assay. Similar results in the third control assay without ATP confirmed the specificity of the RNA aptamer for ADP. Figure S4. In vivo ADP sensor assay. A Preliminary verification for estimating the expression of ADP sensor and monitoring its fluorescence in non-recombinant (DH5α) and recombinant (SSL141) E. coli strains, where the results indicated positive increase in the fluorescence with SSL141 at pH 7.4. B Reproducibility of the assay was confirmed in the recombinant E. coli, in biological triplicates. C PCC 6803 WT and recombinant (SSL142) strains were grown under atmospheric CO2 conditions. ADP sensor expression and working was monitored in both the strains at the optimized reaction pH 4, where SSL142 exhibited increase in the fluorescence over WT. Figure S5. Standard curve for protein contents estimation. Standard curve was obtained by performing Bradford’s protein assay. Bovine serum albumin (BSA) was used as standard. Protein concentration of unknown sample was calculated from linear regression equation y = 0.1286x – 0.0095 obtained from the above plot. Protocol S1. Table S1. Assay reaction mixture. Table S2. Comparison of current RuBisCO activity with previously reported activities. Table S3. Protocol for in vitro transcription and purification of RNA. Table S4. Statistical analysis for determining reproducibility of the assay. All the assays were repeated in biological triplicates to determine their reproducibility. The results indicated that for all the assays, p &gt; 0.05, indicating no significant difference between the activity values obtained while reproducing the assays. This confirmed the assay reproducibility. Sequence S1. Spinach based ADP sensor nucleotide sequence for in vitro studies. Sequence S2. Spinach based ADP sensor nucleotide sequence for in vivo studies.