This research was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research Award Number DE-SC0019363, the NSF CAREER Award CBET-1751840, The Pew Charitable Trusts, and the Camille Dreyfus Teacher-Scholar Award.

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The authors have applied for several patents for the optogenetic circuits and methods described in this article.

Dynamic control has long been applied to improve yields for metabolic engineering and recombinant protein production4. Shifts in enzymatic expression are most typically implemented using chemical inducers such as IPTG21, galactose22, and tetracycline23, but have also been mediated using process conditions such as temperature and pH. Optogenetic control of gene expression eliminates the need for changes to fermentation paramete...

Optogenetics, the control of biological processes with light-responsive proteins, offers a new strategy to dynamically control microbial fermentations for chemical and protein production1,2. The burden of engineered metabolic pathways and the toxicity of some intermediates and products often impairs cell growth3. Such stresses can lead to poor biomass accumulation and reduced productivity3. This challenge can be addressed by temporally dividing fermentations into a growth and production phase, which devote metabolic resources to biomass accumulation or product synthesis respectively4. We recently showed that the transition from growth to production in this two-phase fermentation can be induced with changes in illumination conditions5,6,7. The high tunability, reversibility, and orthogonality of light inputs8 offer unique advantages to light-controlled fermentations that are difficult or impossible to replicate with chemical inducers used in dynamical control of conventional two-phase fermentations4,9,10,11.
The blue-light responsive EL222 protein derived from Erythrobacter litoralis has been used to develop several optogenetic circuits for metabolic engineering in Saccharomyces cerevisiae5,7,12,13. EL222 contains a light-oxygen-voltage sensor (LOV) domain that undergoes a conformational shift upon blue light activation (465 nm), which allows it to bind to its cognate DNA sequence (C120)13. Fusing EL222 to the viral VP16 activation domain (VP16-EL222) results in a blue-light responsive transcription factor that can reversibly activate gene expression in S. cerevisiae7 and other organisms14 from the synthetic promoter PC120. Several circuits based on EL222 have been developed and used for chemical production in S. cerevisiae, such as the basic light-activated OptoEXP system7, in which the gene of interest is directly expressed from PC120&#160;(Figure 1A). However, concerns of light penetration at the high cell densities typically encountered in the production phase of fermentations motivated us to develop inverted circuits that are induced in the dark, such as the OptoINVRT and OptoQ-INVRT circuits (Figure 1B)5,7,13. These systems harness the galactose (GAL) or quinic acid (Q) regulons from S. cerevisiae and N. crassa, respectively, controlling their corresponding repressors (GAL80 and QS) with VP16-EL222, to repress gene expression in the light and strongly induce it in the dark. Combining OptoEXP and OptoINVRT circuits results in bidirectional control of gene expression, enabling two-phase fermentations in which the growth phase is induced with blue light, and the production phase with darkness (Figure 2A)5,7.
Using light instead of darkness to induce gene expression during the production phase would greatly expand the capabilities of optogenetic controls but would also require overcoming the light penetration limitations of the high cell densities typically encountered in this phase of fermentation. To this end, we have developed circuits, known as OptoAMP and OptoQ-AMP, that amplify the transcriptional response to blue light stimulation. These circuits use wild-type or hypersensitive mutants of VP16-EL222 to control production of&#160;the transcriptional activators Gal4p or&#160;QF2 of the GAL or Q regulons, respectively, achieving enhanced sensitivity and stronger gene expression with light12,13&#160;(Figure 1C). OptoAMP circuits can achieve complete and homogeneous light induction in 5 L bioreactors at an optical density (measured at 600 nm; OD600) values of at least 40 with only ~0.35% of illumination (5% light dose on only ~7% of the bulk surface). This demonstrates a higher degree of sensitivity compared to OptoEXP, which requires close to 100% illumination12. The ability to effectively induce gene expression with light at high cell densities opens new opportunities for dynamical control of fermentations. This includes operating fermentations in more than two temporal phases, such as three-phase fermentations, in which growth, induction, and production phases are established with unique light schedules to optimize chemical production (Figure 2B)12.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63269/63269fig1v2.jpg" /> 
Figure 1: Optogenetic circuits for dynamic control of S. cerevisiae. The OptoEXP, OptoINVRT, and OptoAMP circuits are based on the light-sensitive VP16-EL222 system. (A) In the OptoEXP circuit, exposure to blue light causes a conformational change and dimerization of VP16-EL222, which exposes a DNA-binding domain and allows for transcription from PC120. The figure has been modified from Zhao et al.7. (B) OptoINVRT circuits harnesses the GAL (shown) or Q regulons to induce expression in the dark. In GAL-based circuits, VP16-EL222 and GAL4 are constitutively expressed, while PC120 drives expression of the GAL80 repressor (in Q-based circuits, GAL4 and GAL80 are replaced by QF2 and QS, respectively, and a synthetic QUAS-containing promoter is used instead of a GAL promoter). In light, Gal80p prevents activation of the gene of interest from PGAL1. In the dark, GAL80 is not expressed and rapidly degraded by fusing it to a constitutive degron domain (small brown domain),&#160;which allows for activation of PGAL1 by Gal4p. The figure has been modified from Zhao et al.5. (C) OptoAMP circuits also use VP16-EL222 to control the GAL (shown) or Q regulons. In these circuits, the GAL80 repressor (or QS) is constitutively expressed and fused to a photo-sensitive degron (small blue domain) ensuring tight repression in the dark. PC120 and a hypersensitive VP16-EL222 mutant control expression of GAL4 (or QF2) with light, which strongly activates PGAL1&#160;(or a QUAS-containing promoter) in the light. GAL-derived circuits can use engineered forms of PGAL1, such as PGAL1-M or PGAL1-S, which have increased activity, as well as wild-type promoters controlled by the GAL regulon (PGAL1, PGAL10, PGAL2, PGAL7). The figure has been modified from Zhao et al.12. <a href="https://www.jove.com/files/ftp_upload/63269/63269fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63269/63269fig02.jpg" /> 
Figure 2: Two- and three-phase fermentations through time. (A) Two-phase fermentations operated with inverted circuits consist of a light-driven growth phase and a dark production phase. In the growth phase, biomass accumulates as the production pathway stays repressed. Upon reaching the desired OD600, cells are shifted to the dark to metabolically adjust before being resuspended in fresh media for the production phase. (B) In a three-phase process, the growth, incubation, and production phases are defined by unique light schedules, which may consist of a dark growth period, pulsed incubation, and fully illuminated production phase. Figure created with Biorender. <a href="https://www.jove.com/files/ftp_upload/63269/63269fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Optogenetic circuits have also been developed for dynamical control of chemical and protein production in E. coli. OptoLAC circuits control the bacterial LacI repressor using the light-responsive pDawn circuit, which is based on the YF1/FixJ two-component system6 (Figure 3). Similar to OptoINVRT5, OptoLAC circuits are designed to repress gene expression in the light and induce it in the dark. Expression levels using OptoLAC circuits can match or exceed those achieved with standard isopropyl &#946;-d-1-thiogalactopyranoside (IPTG) induction, thus maintaining the strength of chemical induction while offering enhanced tunability and reversibility6. Therefore, OptoLAC circuits enable effective optogenetic control for metabolic engineering in E. coli.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63269/63269fig03.jpg" /> 
Figure 3: OptoLAC circuits for dynamic control of E. coli. The OptoLAC circuits adapt the pDawn system and lac operon to achieve activation in the dark and repression in the light. In the dark, YF1 phosphorylates FixJ, which then activates the PFixK2 promoter to express the cI repressor. The cI repressor prevents expression of the lacI repressor&#160;from the PR promoter, which permits transcription of the gene of interest from a lacO-containing promoter. Conversely, blue light reduces YF1 net kinase activity, reversing FixJ phosphorylation and thus cI expression, which derepresses expression of lacI and prevents expression from the lacO-containing promoter. The figure has been modified from Lalwani et al.6. <a href="https://www.jove.com/files/ftp_upload/63269/63269fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
We describe here the basic protocols for light-controlled fermentations of S. cerevisiae and E. coli for chemical or protein production. For both yeast and bacteria, we first focus on fermentations with a light-driven growth phase and a darkness-induced production phase enabled by OptoINVRT and OptoLAC circuits. Subsequently, we describe a protocol for a three-phase (growth, induction, production) light-controlled fermentation enabled by OptoAMP circuits. Furthermore, we describe how to scale up optogenetically controlled fermentations from microplates to lab-scale bioreactors. With this protocol, we aim to provide a complete and easily reproducible guide for performing light-controlled fermentations for chemical or protein production.

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		<tr>
			<td>Light-controlled chemical production using S. cerevisiae</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well culture plate</td>
			<td>USA Scientific</td>
			<td>CC7672-7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar powder</td>
			<td>Thermo Fisher Scientific</td>
			<td>303991049</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynolds</td>
			<td>B004NG90YO</td>
			<td></td>
		</tr>
		<tr>
			<td>BioSpectrometer with &#956;cuvette</td>
			<td>Eppendorf</td>
			<td>6135000923</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue LED panel</td>
			<td>HQRP</td>
			<td>884667106091218</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-L439 OptoINVRT7 Plasmid</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>See Reference 1</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Thermo Fisher Scientific</td>
			<td>501879892 (G8270-5KG)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002403</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5510</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>Yamato Scientific America</td>
			<td>SOU-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Celltreat</td>
			<td>229656</td>
			<td></td>
		</tr>
		<tr>
			<td>PmeI</td>
			<td>New England Biolabs</td>
			<td>R0560L</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum meter</td>
			<td>Apogee Instruments</td>
			<td>MQ-510</td>
			<td></td>
		</tr>
		<tr>
			<td>Replica-plating device</td>
			<td>Thomas Scientific</td>
			<td>F37848-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Replica-plating pads</td>
			<td>Sunrise Science Products</td>
			<td>3005-012</td>
			<td></td>
		</tr>
		<tr>
			<td>SC-His powder</td>
			<td>Sunrise Science Products</td>
			<td>1303-030</td>
			<td></td>
		</tr>
		<tr>
			<td>SC Complete powder</td>
			<td>Sunrise Science Products</td>
			<td>1459-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile sealing film</td>
			<td>Excel Scientific</td>
			<td>STR-SEAL-PLT</td>
			<td></td>
		</tr>
		<tr>
			<td>YPD agar plates</td>
			<td>VWR</td>
			<td>100217-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>Thermo Fisher Scientific</td>
			<td>R25005</td>
			<td></td>
		</tr>
		<tr>
			<td>Light-controlled protein production using E. coli</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6X SDS Sample Buffer</td>
			<td>Cepham Life Sciences</td>
			<td>10502</td>
			<td></td>
		</tr>
		<tr>
			<td>12% Acrylamide protein gels</td>
			<td>Thermo Fisher Scientific</td>
			<td>NP0341BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well culture plate</td>
			<td>USA Scientific</td>
			<td>CC7672-7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynolds</td>
			<td>B004NG90YO</td>
			<td></td>
		</tr>
		<tr>
			<td>BioSpectrometer with &#956;cuvette</td>
			<td>Eppendorf</td>
			<td>6135000923</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue LED panel</td>
			<td>HQRP</td>
			<td>884667106091218</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue G-250</td>
			<td>Thermo Fisher Scientific</td>
			<td>20279</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis cell</td>
			<td>Bio-Rad</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis power supply</td>
			<td>Bio-Rad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth (Miller)</td>
			<td>Fisher Scientific</td>
			<td>BP97235</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002403</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5510</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Thomas Scientific</td>
			<td>SX0425-1</td>
			<td></td>
		</tr>
		<tr>
			<td>OptoLAC plasmids</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>See Reference 2</td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>Yamato Scientific America</td>
			<td>SOU-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Celltreat</td>
			<td>229656</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum meter</td>
			<td>Apogee Instruments</td>
			<td>MQ-510</td>
			<td></td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>15544034</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5382000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP1521</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-phase fermentation using S. cerevisiae</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Same materials as &#34;Light-controlled chemical production using S. cerevisiae&#34; protocol plus the following:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-L580 OptoAMP4 Plasmid</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>See Reference 10</td>
		</tr>
		<tr>
			<td>Chemical production in a light-controlled bioreactor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynolds</td>
			<td>B004NG90YO</td>
			<td></td>
		</tr>
		<tr>
			<td>Antifoam</td>
			<td>Sigma-Aldrich</td>
			<td>A8311</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioreactor with control station</td>
			<td>Eppendorf</td>
			<td>B120110001</td>
			<td></td>
		</tr>
		<tr>
			<td>BioSpectrometer with &#956;cuvette</td>
			<td>Eppendorf</td>
			<td>6135000923</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>VWR Scientific</td>
			<td>89501-620 (CS)</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue LED panel</td>
			<td>HQRP</td>
			<td>884667106091218</td>
			<td></td>
		</tr>
		<tr>
			<td>BPT tubing</td>
			<td>Fisher Scientific</td>
			<td>14-170-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Thermo Fisher Scientific</td>
			<td>501879892 (G8270-5KG)</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Fisher Scientific</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>M9 Minimal Salts</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1374401</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002403</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5510</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4OH Solution</td>
			<td>Sigma-Aldrich</td>
			<td>I0503-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>Yamato Scientific America</td>
			<td>SOU-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum meter</td>
			<td>Apogee Instruments</td>
			<td>MQ-510</td>
			<td></td>
		</tr>
		<tr>
			<td>SC Complete powder</td>
			<td>Sunrise Science Products</td>
			<td>1459-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

light-controlled fermentations for microbial chemical and protein production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic regulation of microbial metabolism has been successfully implemented to produce a variety of products, including biofuels, bulk chemicals, proteins, and natural products5,6,7,12,13. Most of these processes are designed for cell growth to occur in the light (when low cell density poses minimal challenges with light penetration), and for production t...

Watch this Scientific Journal Video about Light-Controlled Fermentations for Microbial Chemical and Protein Production at JoVE.com

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

light-controlled-fermentations-for-microbial-chemical-protein

Research

JoVE Journal

Bioengineering

3.9K Views.  Princeton University. Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Video: Light-Controlled Fermentations for Microbial Chemical and Protein Production

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...