Name: light-controlled fermentations for microbial chemical and protein production
Uploaded: 2022-03-22T14:00:00
Description: Watch this Scientific Journal Video about Light-Controlled Fermentations for Microbial Chemical and Protein Production at JoVE.com

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.

1. Light-controlled chemical production using the S. cerevisiae OptoINVRT7 circuit
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	<li>Strain construction
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		<li>Obtain a strain with his3 auxotrophy, as this marker is necessary for most existing OptoINVRT plasmids5. If seeking optogenetic regulation of a gene that is native to S. cerevisiae, construct a strain in which any endogenous copy of the gene is deleted.</li>
		<li>Linearize the plasmid containing the OptoINVRT7 circuit, such as ...

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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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<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, ...

Optogenetics has been used to achieve higher titers of several chemicals and proteins compared to typical inducers. Using this protocol, others can enhance their own processes with light-based control. Optogenetics control is noninvasive, highly tuneable, and reversible.These qualities allow for a streamlined optimization of microbial processes and even open unique opportunities, like three-phase fermentations and multi-chromatic control. For any new chemical, the optimal light conditions will differ. So if low production is observed using the parameters in this protocol, different light conditions should be tested.Begin by obtaining a Saccharomyces cerevisiae strain with HIS3 oxytrophy, a marker necessary for OptoINVRT plasmids. If seeking to control a gene native to Saccharomyces cerevisiae, ensure that the endogenous copy of the gene is deleted before proceeding. To begin the strain construction, linearize the plasmid containing the OptoINVRT7 circuit, such as EZL439.If using EZL439, which contains the components to repress the PIGA one promoter in the light and activate it in the dark, linearize at the Pme1 restriction site. Using standard lithium acetate transformation methods, integrate the plasmid into a HIS3 auxotrophic strain. Following the transformation, centrifuge the cells at 150 G's for one minute.Gently resuspend the cells in 200 microliters of fresh SC-HIS medium. Plate the entire cell volume onto an SC-HIS agar plate and incubate at 30 degree Celsius for two to three days until colonies appear. Prepare competent cells using standard lithium acetate transformation protocols.Transform the cells with the plasmid containing the genes that you wish to optogenetically control downstream of either the PIGA1M or PIGA1S promoters. After transformation, centrifuge the culture at 150 G's for one minute and gently resuspend the cell pellet in 200 microliters of fresh SC drop-out medium. Plate the entire cell volume on a yeast extract peptone dextrose agar plate, if integrating into Delta sites or an SC drop-out plate if transforming with a plasmid containing a selection marker.Incubate the cells at 30 degree Celsius for 16 hours under constant blue light to keep the optogenetically controlled gene repressed. Use any light source of the wavelength of 465 nanometers and place an LED panel approximately 40 centimeters above the plate such that the light intensity is approximately 80 to 110 micromoles per square meter per second. Measure the intensity using a quantum meter.If integrating into Delta sites, replicate the plate onto yeast extract peptone dextrose plates containing a range of Zeocin concentrations between 400 micrograms per milliliter and 1, 200 micrograms per milliliter to select for a variety of integration copy numbers. Incubate the replica plates at 30 degree Celsius under constant or pulsed blue light for two to three days until the colonies appear. To perform the preliminary screening, select eight colonies from each plate and use them to inoculate one milliliter of SC-HIS medium supplemented with 2%glucose in individual wells of a 24 well plate.Grow the cells overnight at 30 degree Celsius shaking at 200 revolutions per minute under constant blue light illumination. The next day, dilute each culture in a fresh SC-HIS medium with 2%glucose to obtain the optical density values ranging from 0.01 to 0.3 at 600 nanometers and grow the cultures in shaking condition 200 revolutions per minute under constant or pulsed light at 30 degrees Celsius until the optical density reaches between two and nine. Then, wrap the plates with aluminum foil, turn off the light panel, and incubate the plates in the dark for four hours at 30 degrees Celsius with 200 revolutions per minute.Centrifuge the cultures in the 24 well plate at 234 G's for five minutes and resuspend cells in one milliliter of fresh synthetic complete drop-out medium with 2%glucose. Seal the plates to prevent evaporation of the desired product using sterile microplate sealing tape. Ferment the sealed plates in the dark for 48 hours at 30 degree Celsius shaking at 200 revolutions per minute.To harvest the fermentations, centrifuge the plates for five minutes at 234 G's and transfer 800 microliters of the supernatant into a 1.5 milliliter microcentrifuge tube. Depending on the chemical of interest, analyze using high performance liquid chromatography, gas chromatography mass spectrometry, or another analytical method using the sample preparation technique best suited for the instrument used. Select eight colonies from each plate and use them to inoculate one milliliter of SC-HIS medium with 2%glucose in individual wells of a 24 well plate.Grow the cells overnight in the dark at 30 degree Celsius with 200 revolutions per minute shaking. The next morning, dilute each culture in a fresh SC-HIS medium with 2%glucose to 0.1 optical density. Wrap the plates in aluminum foil to prevent exposure to light and grow the culture in the dark at 30 degree Celsius with 200 revolutions per minute shaking until they reach an optical density of three.Then, incubate the plates under pulsed light for 12 hours at 30 degree Celsius with 200 revolutions per minute shaking. Centrifuge the cultures at 234 G's for five minutes and resuspend in fresh SC-HIS medium with 2%glucose. Seal the plates to prevent evaporation of the desired product using sterile microplate sealing tape.Ferment the sealed plates in light for 48 hours at 30 degrees Celsius shaking at 200 revolutions per minute. After performing optogenetic fermentations, chemical production was tested at a range of cell densities at the time of induction with two circuits. The OptoINVRT7 circuit demonstrated high titers of lactic acid and isobutanol with an optimal cell density of induction value of 7.0 and 8.75 compared to the OptoINVRT1 and 2 circuits.Fermentations using the OptoAMP circuits were studied in three phases defined by different light duty cycles. Lactic acid production can be optimized using a pulsed growth phase and fully illuminated induction and production phases. Isobutanol production was optimized using a pulsed growth phase, fully illuminated induction phase, and pulsed production phase.Whereas naringenin biosynthesis was best optimized using a pulsed growth fully illuminated induction and dark production phase. The OptoLAC system has been used in E.Coli to produce transcription factor FDER at titers that are comparable to or higher than those achieved using chemical induction with IPDG. OptoLAC circuits have been applied to produce mevalonate and the production exceeds titers achieved using IPDG induction.Mevalonate production at the bioreactor level demonstrates the scalability and tunability of optogenetic regulation for chemical and protein production in bacteria beyond microplate levels. It's important to ensure that the light intensity is in the proper range for the illuminated steps and that light contamination is avoided for steps that require darkness. This technique paved the way to achieve record titers of isobutanol in yeast, stabilized consortia populations with light, and even control metabolic flux using synthetic light controlled organelles.

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