Papers by Poonam Srivastava
Transformants were grown in unsupplemented MOPS glucose medium. . Western blot analysis of Lrp ac... more Transformants were grown in unsupplemented MOPS glucose medium. . Western blot analysis of Lrp accumulation (Eco, Lrp; Pmi, Lrp; Vch, Lrp; pCC1, vector control) using polyclonal antiserum raised against Lrp. The arrow indicates the direction of electrophoresis. . P(B), P(C) and P(D) activity were measured via ONPG hydrolysis, and plotted . culture density to ensure that the cultures were in balanced growth. The Lrp orthologs used are from (triangles) and (squares), as well as the positive control (circles) and the vector control (diamonds). . Isoleucine, Leucine and Valine was added to the medium ("+Leu") for experiments depicted in the lower panels: (E), (F) and (G). The correlation coefficients for the least-squares fits to the data were all at least 0.97.<b>Copyright information:</b>Taken from "Limited functional conservation of a global regulator among related bacterial genera: Lrp in , and "http://www.biomedcentral.com/1471-2180/8/60BMC Microbiol...
Artificial metalloenzymes (ArMs) offer new opportunities to improve catalytic selectivity and eff... more Artificial metalloenzymes (ArMs) offer new opportunities to improve catalytic selectivity and efficiency. They are a class of catalysts that result from incorporation of an organometallic catalyst precursor within a host protein. This combination exploits the reactivity of the transition metal catalysts while taking advantage of the selectivity and adaptability of proteins. We have demonstrated formation of ArMs through strain promoted azide-alkyne cycloaddition (ChemBioChem 2014). It is a ‘click’ bioconjugation approach, where a covalent link is formed between metal complex and protein through cycloaddition of a strained alkyne linker and genetically encoded p-azidophenyl alanine on the protein scaffold. Several scaffold proteins and cofactor components were explored to demonstrate versatility of this method. Extensive biophysical characterization of these ArMs was also done by mass spectrophotometry, fluorescence spectroscopy and X-ray crystallography. Scaffold proteins were engin...
The data-driven design of protein sequences with desired function is challenged by the absence of... more The data-driven design of protein sequences with desired function is challenged by the absence of good theoretical models for the sequence-function mapping and the vast size of protein sequence space. Deep generative models have demonstrated success in learning the sequence to function relationship over natural training data and sampling from this distribution to design synthetic sequences with engineered functionality. We introduce a deep generative model termed the Protein Transformer Variational AutoEncoder (ProT-VAE) that furnishes an accurate, generative, fast, and transferable model of the sequence-function relationship for data-driven protein engineering by blending the merits of variational autoencoders to learn interpretable, low-dimensional latent embeddings and fully generative decoding for conditional sequence design with the expressive, alignment-free featurization offered by transformers. The model sandwiches a lightweight, task-specific variational autoencoder between...
Sequences upstream of , and orthologs. In each case, the sequence ends with the initiation codon.... more Sequences upstream of , and orthologs. In each case, the sequence ends with the initiation codon. Lrp-binding sites and the transcriptional +1 position are known for -12 [112]. Demonstrated Lrp binding sites are in , and the -35 and -10 sequences inferred from the known +1 position (for ) are boxed. Putative binding sites, predicted by the PRODORIC virtual footprinter [102] are shaded, and the match scores for predicted sites are shown to the right. For P, one of the predicted sites overlaps an actual site, and gives a particularly high match score, though an overlapping actual site in Pdoes not. has two nearly-tandem copies of the operon on chromosome I. The 5'-most isozyme ("Vch1", locus tag Vc2373) is 43% identical to Eco , while the 3'-most isozyme ("Vch2", Vc2376) is 73% identical to Eco in amino acid sequence. Samples were isolated at an ODof 0.3 (log), as well as 1 h after linear growth stopped (stationary), from and wild-type and cultures growing ...
Ng standard errors. At least four points from each of two independent experiments were used to ge... more Ng standard errors. At least four points from each of two independent experiments were used to generate each plotted value. Conditions were MOPS-glucose minimal medium, supplemented as described in Methods, in logarithmic (MinLog) or stationary phase (MinSta), or MOPS glucose defined rich medium in those growth phases (RchLog, RchSta). . Direct comparison of mRNA levels.. A baseline amount of total RNA (from a mid-log phase culture in MOPS glucose plus nicotinate) was mixed with varying amounts of test RNA (all from log-phase cultures) from glucose (open circle) or rich (closed circle) cultures. The mixes were used as template for simultaneous amplification with three primer pairs. If the test cDNA preparation has the same proportion of cDNA as the reference pool, the detected amount of cDNA should rise with a slope of 1.0 (actual . detected, based on the varied amounts of test cDNA added); this is shown as a dotted line in each panel.<b>Copyright information:</b>Taken f...
Gene expression was assessed by two-color microarray analysis as described in Methods. The pie ch... more Gene expression was assessed by two-color microarray analysis as described in Methods. The pie chart represents the relative distribution of genes significantly responsive to the Lrp that are also significantly responsive to the other Lrp orthologs. Details of the statistical analysis of these data are in Methods, and the gene-specific results are available [see Additional file ].<b>Copyright information:</b>Taken from "Limited functional conservation of a global regulator among related bacterial genera: Lrp in , and "http://www.biomedcentral.com/1471-2180/8/60BMC Microbiology 2008;8():60-60.Published online 11 Apr 2008PMCID:PMC2374795.
Open symbols refer to growth in MOPS glucose plus required supplements (nicotinate, panthothenate... more Open symbols refer to growth in MOPS glucose plus required supplements (nicotinate, panthothenate and thiamine; see Methods), while closed symbols represent growth in MOPS glucose defined-rich medium. . growth rates. The values shown are the specific growth rate constants, , calculated as ln2/(doubling time, in h). For comparison, values of 0.5, 1, and 2 correspond respectively to doubling times of 83, 42, and 21 min. The rich medium results are clustered and therefore not labeled; for the minimal medium, the abbreviations used are Eco (), Pmi (), and Vch (). The diagonal line shows where points should fall if mutation has no effect on the growth rate in these media. . Complementation of the low growth rate in the glucose minimal medium described in (A). The dashed lines indicate growth data for the mutant (open circles; 193 min doubling time) and the mutant bearing the vector control (gray circles; 191 min). Remaining lines show the WT (closed circles; 66 min); and the mutant beari...
ChemBioChem, 2015
A bicyclo[6,1,0]nonyne-substituted 9-mesityl-10-methyl-acridinium cofactor was prepared and coval... more A bicyclo[6,1,0]nonyne-substituted 9-mesityl-10-methyl-acridinium cofactor was prepared and covalently linked to a prolyl oligopeptidase scaffold containing a genetically encoded 4-azido-Lphenylalanine residue in its active site. The resulting artificial enzyme catalyzed sulfoxidation when irradiated with visible light in the presence of air. This reaction proceeds via initial electron abstraction from the sulfide within the enzyme active site, and the protein scaffold extended the fluorescence lifetime of the acridium cofactor. The mode of sulfide activation and placement of 5 in POP-ZA 4-5 make this artificial enzyme a promising platform for developing selective photocatalytic transformations. Keywords artificial enzyme; photocatalyst; oxygenase; prolyl oligopeptidase; unnatural amino acid Chemists have long sought to design catalysts that mimic the ability of natural photosynthetic systems to harvest visible light photons and use the energy from photon absorption to drive chemical reactions. [1] The protein complexes that evolved to accomplish these tasks, photosystems II and I, catalyze light-driven water oxidation and transmembrane electron transport, respectively. [2] Photosynthetic organisms use the electrons transported by photosystem I (PSI) to reduce NADP + to NADPH, but a number of biohybrid systems capable of harnessing these electrons to reduce protons to H 2 for solar fuels applications have been developed by linking PSI to synthetic catalysts. [3] These systems show that the unique photophysical properties imparted to natural chromophores embedded within the PSI reaction center protein scaffolds [4] can be exploited for non-natural chemical reactions. Recently, an alternative biohybrid catalyst for light-driven proton reduction was created via bioconjugation of a hydrogen-evolving diiron complex to a protein scaffold lacking any bound chromaphores. [5] The photosensitizer [Ru(bpy) 3 ] 2+ was added to a solution of the biohybrid to enable intermolecular electron transfer to the diiron complex upon irradiation with visible light and catalytic proton reduction in the presence of ascorbate. Prior to this work, Gray [6] and Cheruzel [7] had demonstrated that electrons supplied to a cytochrome P450 BM3 variant by a covalently linked [Ru(bpy) 3 ] 2+ derivative upon irradiation with
Molecular Biotechnology, 2005
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, that ... more Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, that has been used as a therapeutic agent in facilitating bone marrow and stem cell transplantation and in other clinical cases like neutropenia. Although biologically active recombinant GM-CSF has been successfully produced in Escherichia coli, the reported levels are extremely poor. In this study we looked into the possible reasons for poor expression and found that protein toxicity coupled with protease-based degradation was the principal reason for low productivity. To overcome this problem we attached a signal sequence, as well as an amino-terminal His-tag fusion to the GM-CSF gene. This combination had a dramatic effect on expression levels, which increased from 0.8 µg/mL in the control to 40 µg/mL. When a larger fusion partner, such as the Maltose-binding protein (MBP-tag), was used the expression levels increased further to 69.5 µg/mL, which along with the MBP-tag represented approx 12% of the total cellular protein.
Biochemical Engineering Journal, 2005
Kinetic modeling of rhIFN-α expression was done in continuous cultures using complex media at dil... more Kinetic modeling of rhIFN-α expression was done in continuous cultures using complex media at dilution rates varying from 0.2 to 0.5h−1. The human IFN-α gene was inserted under the T7 promoter in BL21 (DE3) codon plus (RIL) cells and induced with 1mM IPTG. Post-induction growth and production profiles were monitored over time. Wash out of the cells was observed only
Applied Microbiology and Biotechnology, 2011
The BioBrick™ paradigm for the assembly of enzymatic pathways is being adopted and becoming a sta... more The BioBrick™ paradigm for the assembly of enzymatic pathways is being adopted and becoming a standard practice in microbial engineering. We present a strategy to adapt the BioBrick™ paradigm to allow the quick assembly of multi-gene pathways into a number of vectors as well as for the quick mobilization of any cloned gene into vectors with different features for gene expression and protein purification. A primary BioBrick™ (BB-eGFP) was developed where the promoter/RBS, multiple cloning sites, optional protein purification affinity tags and reporter gene were all separated into discrete regions by additional restriction enzymes. This primary BB-eGFP then served as the template for additional BioBrick™ vectors with different origins of replication, antibiotic resistances, inducible promoters (arabinose, IPTG or anhydrotetracycline), N- or C-terminal Histidine tags with thrombin cleavage, a LacZα reporter gene and an additional origin of mobility (oriT). All developed BioBricks™ and BioBrick™ compatible vectors were shown to be functional by measuring reporter gene expression. Lastly, a C(30) carotenoid pathway was assembled as a model enzymatic pathway to demonstrate in vivo functionality and compatibility of this engineered vector system.
BMC Microbiology, 2008
Background Bacterial genome sequences are being determined rapidly, but few species are physiolog... more Background Bacterial genome sequences are being determined rapidly, but few species are physiologically well characterized. Predicting regulation from genome sequences usually involves extrapolation from better-studied bacteria, using the hypothesis that a conserved regulator, conserved target gene, and predicted regulator-binding site in the target promoter imply conserved regulation between the two species. However many compared organisms are ecologically and physiologically diverse, and the limits of extrapolation have not been well tested. In E. coli K-12 the leucine-responsive regulatory protein (Lrp) affects expression of ~400 genes. Proteus mirabilis and Vibrio cholerae have highly-conserved lrp orthologs (98% and 92% identity to E. coli lrp). The functional equivalence of Lrp from these related species was assessed. Results Heterologous Lrp regulated gltB, livK and lrp transcriptional fusions in an E. coli background in the same general way as the native Lrp, though with sig...
Tetrahedron, 2014
New catalysts for non-directed hydrocarbon functionalization have great potential in organic synt... more New catalysts for non-directed hydrocarbon functionalization have great potential in organic synthesis. We hypothesized that incorporating a Mn-terpyridine cofactor into a protein scaffold would lead to artificial metalloenzymes (ArMs) in which the selectivity of the Mn cofactor could be controlled by the protein scaffold. We designed and synthesized a maleimide-substituted Mnterpyridine cofactor and demonstrated that this cofactor could be incorporated into two different scaffold proteins to generate the desired ArMs. The structure and reactivity of one of these ArMs was explored, and the broad oxygenation capability of the Mn-terpyridine catalyst was maintained, providing a robust platform for optimization of ArMs for selective hydrocarbon functionalization.
1. Purpose This BioBricks Foundation Request for Comments (BBF RFC) provides instructions on how ... more 1. Purpose This BioBricks Foundation Request for Comments (BBF RFC) provides instructions on how to automatically generate a model of any BioBrick sequence. These models can be used in computer simulations of the dynamic behavior of all molecular components of the BioBrick. BBSF RFC 40 does not update or replace any earlier BBF RFC. Modeling tools can play an important role in synthetic biology the same way modeling helps in aircraft or architecture design: simulations can quickly provide a clear picture of how different components influence the behavior of the whole [1,2]. With SynBioSS Designer synthetic biologists and engineers can quickly construct models of synthetic biological systems. Users can enter DNA sequences, including BioBricks, and Designer returns a network of reactions that model all the steps of the molecular biology dogma. With these models, users can study the dynamic behavior of the synthetic construct and make the necessary connection between DNA sequence and b...
This BioBricks Foundation Request for Comments (BBF RFC) provides instructions on how to automati... more This BioBricks Foundation Request for Comments (BBF RFC) provides instructions on how to automatically generate a model of any BioBrick sequence. These models can be used in computer simulations of the dynamic behavior of all molecular components of the BioBrick. 2. Relation to other BBF RFCs BBSF RFC 40 does not update or replace any earlier BBF RFC.
Journal of Chemical & Engineering Data, 2014
The tetracycline operon is an important gene network component, commonly used in synthetic biolog... more The tetracycline operon is an important gene network component, commonly used in synthetic biology applications because of its switch-like character. At the heart of this system is the highly specific interaction of the tet repressor protein (TetR) with its cognate DNA sequence (tetO). TetR binding on tetO practically stops expression of genes downstream of tetO by excluding RNA polymerase from binding the promoter and initiating transcription. Mutating the tetO sequence alters the strength of TetR−tetO binding and thus provides a tool to synthetic biologists to manipulate gene expression levels. We employ molecular dynamics (MD) simulations coupled with the free energy perturbation method to investigate the binding affinity of TetR to different tetO mutants. We also carry out in vivo tests in Escherichia coli for a series of promoters based on these mutants. We obtain reasonable agreement between experimental green fluorescent protein (GFP) repression levels and binding free energy differences computed from molecular simulations. In all cases, the wildtype tetO sequence yields the strongest TetR binding, which is observed both experimentally, in terms of GFP levels, and in simulation, in terms of free energy changes. Two of the four tetO mutants we tested yield relatively strong binding, whereas the other two mutants tend to be significantly weaker. The clustering and relative ranking of this subset of tetO mutants is generally consistent between our own experimental data, previous experiments with different systems and the free energy changes computed from our simulations. Overall, this work offers insights into an important synthetic biological system and demonstrates the potential, as well as limitations of molecular simulations to quantitatively explain biologically relevant behavior.
Nature chemistry, 2018
Random mutagenesis has the potential to optimize the efficiency and selectivity of protein cataly... more Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. ...
Nature Communications, 2015
Artificial metalloenzymes (ArMs) formed by incorporating synthetic metal catalysts into protein s... more Artificial metalloenzymes (ArMs) formed by incorporating synthetic metal catalysts into protein scaffolds have the potential to impart to chemical reactions selectivity that would be difficult to achieve using metal catalysts alone. In this work, we covalently link an alkynesubstituted dirhodium catalyst to a prolyl oligopeptidase containing a genetically encoded L-4-azidophenylalanine residue to create an ArM that catalyses olefin cyclopropanation. Scaffold mutagenesis is then used to improve the enantioselectivity of this reaction, and cyclopropanation of a range of styrenes and donor-acceptor carbene precursors were accepted. The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium-carbene intermediates with water. This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction. Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.
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Papers by Poonam Srivastava