An official website of the United States government
The function, structure, and mechanical properties of protein materials make them well-suited for a range of applications such as biosensors and biomaterials. Unlike in traditional polymer synthesis, their sequences are defined and, in the case of recombinant proteins, dictated by the chosen DNA sequence. As DNA synthesis has rapidly progressed over the past twenty years, the limiting bottleneck in protein materials development is the empirical optimization of protein expression. Herein, a low-cost, automated, high-throughput, combinatorial protein expression platform is developed to test permutations of DNA vectors and Escherichia coli ( E. coli ) strains in a 96-well plate format. Growth and expression are monitored with optical density at 600 nm (OD 600 ) to measure growth, Bradford assays to establish the total protein concentration, and dot blot assays to determine the concentration of the protein of interest. With an eye toward accessibility for researchers without suites of biosynthetic equipment, automated camera-based assays are validated for the OD 600 assay, via turbidimetry, and the Bradford assay, via colorimetry. High-yield expression conditions can be determined within a week. Notably, in several cases, previously un-expressible proteins are expressed successfully in viable yields. Collectively, an efficient approach to overcoming long-running synthesis challenges in protein materials development is established, which will expedite materials innovation.
more »
« less
Growth of microorganisms in an interfacially driven space bioreactor analog
Abstract Fluid bioreactors in microgravity environments may utilize alternative methods of containment and mixing. The ring-sheared drop (RSD) is a containerless mixing device which functions in microgravity using surface tension for containment and mixes through interfacially-driven flow. To assess the feasibility of using interfacially driven flow devices, such as the RSD, as bioreactors, Escherichia coli growth and recombinant protein expression were analyzed in a ground-based analog of the RSD called the knife edge surface viscometer (KEV). Results demonstrated that the KEV can facilitate the growth of E. coli and that growth rate increases logarithmically with increasing knife edge rotation rate, similar to the standard growth method on Earth (orbital shaker). Furthermore, the KEV was shown to be viable for supporting recombinant protein expression in E. coli at levels comparable to those achieved using standard growth methods.
more »
« less
- Award ID(s):
- 1929134
- PAR ID:
- 10280754
- Date Published:
- Journal Name:
- npj Microgravity
- Volume:
- 6
- Issue:
- 1
- ISSN:
- 2373-8065
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Fluid interfaces significantly influence the dynamics of protein solutions, effects that can be isolated by performing experiments in microgravity, greatly reducing the amount of solid boundaries present, allowing air-liquid interfaces to become dominant. This investigation examined the effects of protein concentration on interfacial shear-induced fibrillization of insulin in microgravity within a containerless biochemical reactor, the ring-sheared drop (RSD), aboard the international space station (ISS). Human insulin was used as a model amyloidogenic protein for studying protein kinetics with applications to in situ pharmaceutical production, tissue engineering, and diseases such as Alzheimer’s, Parkinson’s, infectious prions, and type 2 diabetes. Experiments investigated three main stages of amyloidogenesis: nucleation studied by seeding native solutions with fibril aggregates, fibrillization quantified using intrinsic fibrillization rate after fitting measured solution intensity to a sigmoidal function, and gelation observed by detection of solidification fronts. Results demonstrated that in surface-dominated amyloidogenic protein solutions: seeding with fibrils induces fibrillization of native protein, intrinsic fibrillization rate is independent of concentration, and that there is a minimum fibril concentration for gelation with gelation rate and rapidity of onset increasing monotonically with increasing protein concentration. These findings matched well with results of previous studies within ground-based analogs.more » « less
-
Recombinant protein production plays an essential role in both biological studies and pharmaceutical production. Escherichia coli is one of the most favorable hosts for this purpose. Although a number of strategies for optimizing protein production have been developed, the effect of gene overexpression on host cell growth has been much less studied. Here, we performed high-throughput tests on the E. coli a complete set of E. coli K-12 ORF archive (ASKA) collection to quantify the effects of overexpressing individual E. coli genes on its growth. The results indicated that overexpressing membrane-associated proteins or proteins with high abundances of branched-chain amino acids tended to impair cell growth, the latter of which could be remedied by amino acid supplementation. Through this study, we expect to provide an index for a fast pre-study estimate of host cell growth in order to choose proper rescuing approaches when working with different proteins.more » « less
-
RationalePurification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI). MethodsA protein standard, His‐Ubq, and two recombinant proteins, His‐SHAN and His‐CS, expressed inEscherichia coliwere immobilized on two immobilized metal affinity systems, Cu–nitriloacetic acid (Cu‐NTA) and Ni‐NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96‐well plate form factor, or analyzed directly from immobilized metal affinity‐coated microscope slides by DESI‐MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis. ResultsSmall proteins (His‐Ubq) and medium proteins (His‐SAHN) could readily be detected from 96‐well plates by direct infusion ESI, or from microscope slides by DESI‐MS after purification on surface from clarifiedE. colicell lysate. Protein oxidation was observed for immobilized proteins on both Cu‐NTA and Ni‐NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His‐SAHN and the methylation product of His‐CS (theobromine to caffeine) were detected. ConclusionsThe immobilization, purification, release and detection of His‐tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI‐MS or ambient DESI‐MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.more » « less
-
Abstract Investigating the evolution ofEscherichia coliin microgravity offers valuable insights into microbial adaptation to extreme environments. Here the effects of simulated microgravity (SµG) on gene expression and genome evolution ofE. coliREL606, a strain evolved terrestrially for 35 years, is explored. The transcriptomic changes for glucose-limited and glucose-replete conditions over 24 h illustrate that SµG increased the expression of genes involved in stress response, biofilm, and metabolism. A greater number of differentially expressed genes related to the general stress response (GSR) and biofilm formation is observed in simulated microgravity cultures under glucose-limited conditions in comparison to glucose-replete conditions. Longer term SµG culture under glucose-limited conditions led to the accumulation of unique mutations when compared to control cultures, particularly in themraZ/fruRintergenic region and theelyC gene, suggesting changes in peptidoglycan and enterobacterial common antigen (ECA) production. These findings highlight the physiological and genomic adaptations ofE. colito microgravity, offering a foundation for future research into the long-term effects of space conditions on bacterial evolution.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s41526-020-0101-4
