Name: Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials (Video) | JoVE Journal
Uploaded: 2017-03-09T15:00:00.000+00:00
Duration: 10 min 28 s
Description: Learn to engineer E. coli for controlling programmable material surfaces using genetic modification and surface functionalization. This method enables dynamic sensing for molecular medicine and environmental monitoring applications.

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5 minutes

The authors gratefully acknowledge support from award FA9550-13-1-0108 from the Air Force Office of Scientific Research of the USA. The authors additionally acknowledge support from award N00014-15-1-2502 from the Office of Naval Research of the USA, funding from the Institute for Critical Technology and Applied Science at Virginia Polytechnic Institute and State University, and from the National Science Foundation Graduate Research Fellowship Program, award number 1607310.

1. Media and Culture Preparation
<ol>
	<li>Prepare lysogeny broth (LB) media by mixing 25 g of LB powder stock with 1 L of deionized (DI) water and autoclaving the solution at 121 °C for 20 min to sterilize.
	<ol>
		<li>To prepare LB plates, add 15 g agar (1.5%) to the LB media before sterilization</li>
		<li>Prepare stock solutions of 1,000x carbenicillin (Cb) in DI water (50 mg/mL).</li>
		<li>If preparing LB media that includes an antibiotic for selection of resistant transformants, wait until the sterilized LB media's temperature is below 60 °C, and then add 1 µL of antibiotic stock for every 1 mL of LB media.</li>
	</ol>
	</li>
	<li>For M9 minimal media (Table 1), prepare separate stock solutions of the following: 5X M9 salts (56.4 g/L), 1 M MgSO4, 1 M CaCl2, 20% glucose, and 2% biotin-free casamino acids.
	<ol>
		<li>In an autoclave safe bottle, combine 20 mL of 5x M9 salts, 200 µL of 1M MgSO4, 10 µL of 1 M CaCl2, 2 mL of 20% glucose, 1 mL of 2% biotin-free casamino acids, and 76.8 mL of DI water.</li>
		<li>Autoclave the solution prepared in step 1.2.1 at 121 °C for 20 min.</li>
	</ol>
	</li>
	<li>Incubate all cultures at 37 °C with agitation at 400 rpm. Incubation times vary depending on experiment and strain, but typically last from 8 to 12 h.</li>
</ol>
2. Generation of Biotin Producing E. coli (Plasmid pKE1-lacI-bioB)
NOTE: The genetic circuit contains two parts: a lacI repressor, driven by a PL,tetO-1 promoter resulting in constitutive expression due to the absence of a tetR repressor protein, as well as a biotin expression system containing the Ptrc-2 promoter followed by a strong ribosome binding site (rbs) driving expression of bioB. All cloning was executed in a commercial, rapidly dividing E. coli strain. The final construct was transformed into E. coli MG1655WT for testing. Primers (Table 2) were purchased commercially.
<ol>
	<li>Isolate bioB gene from E. coli genome by performing whole-cell polymerase chain reaction (PCR):
	<ol>
		<li>Grow E. coli MG 1655WT cells overnight at 37 °C.</li>
		<li>In a 0.2 mL PCR tube, combine 1 µL of the overnight culture prepared in step 2.1.1 with 9 µL of sterile DI water.</li>
		<li>Incubate the tube at 95 °C for 5 min in a thermocycler and immediately transfer the tube to a -80 °C freezer for 10 min to lyse the cells. This enables genomic DNA to serve as a PCR template.</li>
		<li>Thaw the solution and add (i) 2.5 µL each of the primers nBioB2-f1 and nBioB2-r, (ii) 5 µL of 5x DNA polymerase buffer, (iii) 0.25 µL of DNA polymerase, (iv) 0.5 µL of dNTP mix, and (v) 6.75 µL of DI water for a total reaction volume of 25 µL (Table 4).</li>
		<li>Strike the side of (i.e., flick) the tube to mix its contents. Next, immediately spin down the tube quickly (~ 2 s) to ensure the sample is at the bottom of the tube.</li>
		<li>Place the tube in a PCR thermocycler and use the program described in Table 3 with an additional step of 3 min at 95 °C at the beginning of the protocol.</li>
		<li>Confirm successful PCR using gel electrophoresis (1.0 - 1.2% agarose in DI water+ ethidium bromide) in Tris base, acetic acid, and EDTA buffer (TAE). The PCR product should be 1,070 basepairs (bp) long.</li>
		<li>Use commercial kits according to manufacturer's instructions to extract DNA fragments from gel from step 2.1.7.</li>
	</ol>
	</li>
	<li>Isolate the Ptrc-2 promoter and the lacI operon from the plasmid pKDL07113 (courtesy of the lab of James Collins at MIT):
	<ol>
		<li>Grow E. coli cells containing the pKDL071 plasmid overnight in LB + Cb at 37 °C.</li>
		<li>Extract the plasmid DNA from the cells using a commercial miniprep kit according to the manufacturer's instructions. The plasmid will serve as a PCR template.</li>
		<li>Follow the PCR protocol from steps 2.1.4. to 2.1.8. with the following modifications:</li>
		<li>Replace lysed cells with the plasmid extract in step 2.2.2.</li>
		<li>Use 2.5 µL each of primers 1-f and 1-r for lacI cassette extraction. Alternately, use 2.5 µL each of primers nBioB1-f and nBioB1-r for Ptrc-2 extraction.</li>
		<li>Perform gel electrophoresis (step 2.1.7) to confirm the PCR products are the lacI cassette and Ptrc-2 promoter site. They should be 1213 bp and 109 bp long, respectively.</li>
	</ol>
	</li>
	<li>Use splicing by overlap extension (SOE) PCR to build the bioB cassette containing Ptrc-2, a synthetic ribosome binding site (RBS) in primer nBioB2-f2, and the bioB gene. The thermocyler PCR program is found in Table 3.</li>
	<li>Insert constructs (PL,tetO-1 + lacI and Ptrc-2 + bioB ) into the pKE1-MCS plasmid vector13 backbone (courtesy of the lab of James Collins at MIT) by digesting the vector and insert with restriction enzymes:
	<ol>
		<li>Extract genes using restriction enzymes and gel electrophoresis. Each reaction contains (i) 5 µL of 10x reaction buffer, (ii) 1 µL of restriction enzyme 1, (iii) 1 µL of restriction enzyme 2, (iv) at least 1 µg of DNA, and (v) DI water to bring the final volume to 50 µL (Table 5). Digest with enzymes AatII and EcoRI for the lacI cassette and with enzymes HindIII and SacII for the bioB cassette.</li>
		<li>After the reactions are assembled, quickly vortex them and briefly centrifuge (~ 2 s) before incubation for 1 h at 37 °C.</li>
		<li>During digestion, prepare gels for electrophoresis according to standard protocols.</li>
		<li>Combine digested DNA with gel electrophoresis 6x loading buffer.</li>
		<li>Use a 2-log ladder to verify location of desired construct, cut DNA fragment from the gel, and use commercial gel extraction kits according to manufacturer's instructions to extract DNA fragments from the gel.</li>
	</ol>
	</li>
	<li>Quantify DNA using spectroscopy and calculate volumes for 0:1, 1:1, and 3:1 molar ratios of insert to vector using equation 1: 
	<img alt="Chemical equilibrium equation, formula for partial molar quantities." src="/files/ftp_upload/55300/equation1.jpg" data-altupdatedat="1788250015000" data-alt="Equation 1"> Equation 1 
	In equation 1, M is the ratio of insert to vector (0, 1, or 3), Xi is the quantity of insert DNA, bpi is the length of the insert in base pairs, bpv is the length of the vector in basepairs, and Xv is the quantity of vector (50 ng).</li>
	<li>Mix ligation reaction by combining (i) 1 µL 10X Ligase Buffer, (ii) 1 µL T4 Ligase, (iii) 50 ng vector DNA, (iv) a mass of insert DNA specific to each reaction, and (v) DI water to 10 µL total reaction volume (Table 6).</li>
	<li>Incubate at room temperature (RT) for 1 h.</li>
	<li>During the ligation incubation in step 2.7, prepare chemically competent cells.
	<ol>
		<li>Aliquot 1 mL of overnight cultures into 1.5 mL centrifuge tubes.</li>
		<li>Centrifuge at 16,200 x g for 1 min.</li>
		<li>Decant supernatant and resuspend the pellet in 200 µL of chilled (on ice) 100 mM CaCl2. Resuspend the pellet by gently pipetting. Do not vortex.</li>
		<li>Place the microcentrifuge tube on ice for 10 min.</li>
		<li>Centrifuge, remove the supernatant, resuspend the pellet in 100 µL of chilled 100 mM CaCl2, and place the tube on ice again.</li>
		<li>Centrifuge the tube a final time and resuspend the pellet in 50 µL of chilled 100 mM CaCl2.</li>
		<li>Place the tube on ice and use the chemically competent cells immediately.</li>
	</ol>
	</li>
	<li>Add 5 µL of ligated DNA, prepared in steps 2.6 and 2.7, to each tube of competent cells, prepared in step 2.8.</li>
	<li>Agitate the tube briefly by striking the side (i.e. flicking) the tubes and then place the tubes on ice for 30 min.</li>
	<li>Heat shock the tubes for 45 s at 42 °C and return the tubes to ice for 2 min.</li>
	<li>Pipette cells onto selective LB agar + antibiotic plates and spread the cells using glass plating beads.</li>
	<li>Incubate the plates overnight at 37 °C.</li>
	<li>The following morning, pick colonies from insert-positive plates (i.e., 1:1 and 3:1 plates). Pick 3 colonies if the 0:1 negative control plate shows no growth, or pick 5-8 colonies if the 0:1 plate has some growth. Use the picked colonies to inoculate 5 mL LB + antibiotic and grow for 8 h at 37 °C with agitation.</li>
	<li>Extract the plasmid DNA from the cells using a miniprep kit, following the manufacturer's instructions. Perform a test cut using restriction enzymes (following the same procedure detailed in step 2.4.1.).</li>
	<li>Identify cells with successful constructs by comparing the length of the digested DNA fragments with the results expected from a correct construct. Cultures carrying successful plasmid constructs may be preserved by mixing equal parts culture with a solution of sterile-filtered, 40 % glycerol (in DI water) and freezing the mixture at -80 °C. 
	NOTE: Transformations of plasmids into other E. coli strains (i.e., MG1655WT) can be accomplished by following the above procedure for making chemically competent cells.</li>
</ol>
3. Cell Characterization: Growth Curve and Dose Response
<ol>
	<li>Grow engineered strains overnight in LB media with an appropriate antibiotic.</li>
	<li>For growth curves, prepare 50 mL of minimal M9 media with and without IPTG (from a 0.5 M stock) and/or DTB (from a 500 µg/mL stock) as follows:
	<ol>
		<li>Prepare 0 ng/mL DTB (0 µL of stock)/ 0 mM IPTG (0 µL of stock).</li>
		<li>Prepare 200 ng/mL DTB (200 µL of stock)/ 0 mM IPTG (0 µL of stock).</li>
		<li>Prepare 0 ng/mL DTB (0 µL of stock)/ 0.5 mM IPTG (50 µL of stock).</li>
		<li>Prepare 200 ng/mL DTB (200 µL of stock)/ 0.5 mM IPTG (50 µL of stock).</li>
		<li>Inoculate with overnight culture at 1:100 in the media specified above.</li>
		<li>In a 96-well plate, aliquot 200 µL of culture, in triplicate, to each well.</li>
		<li>Measure OD600 every 5 min over 24 h with continuous shaking and incubation at 37 °C in plate reader.</li>
	</ol>
	</li>
	<li>For dose response studies, replace the bioB gene in pKE1-lacI-bioB with mCherry (a red fluorescent protein) for real-time optical quantification.</li>
	<li>Add varying amounts of IPTG ranging from 0.1 mM to 5 mM to induce expression in LB media.</li>
	<li>Inoculate with overnight culture at a dilution of 1:100.</li>
	<li>Measure fluorescence every 30 min for 15 h.</li>
</ol>
4. Inducing Biotin Production from Engineered Cells and Supernatant Preparation
<ol>
	<li>Grow pKE1-lacI-bioB in the E. coli MG1655 strain overnight in LB media.</li>
	<li>Supplement M9 media with DTB ranging from 30 to 200 ng/mL.</li>
	<li>Add 0.5 mM IPTG to induce biotin synthesis.</li>
	<li>Inoculate supplemented, biotin-free minimal M9 media with overnight culture at 1:100 dilution.</li>
	<li>After 24 h of growth, centrifuge cells and collect the biotin-enriched supernatant.</li>
	<li>Measure biotin-enriched supernatant with the indirect control and the direct control functionalized surfaces. Use the supernatant in place of the biotin sample in steps 5.23 and 6.12, respectively.</li>
</ol>
5. Indirect Control Scheme Functionalized Surface Preparation
<ol>
	<li>Prepare the following solutions.
	<ol>
		<li>Prepare an SMCC solution consisting of 20 mg/mL of succinimidyl trans-4-(maleimidylmethyl) cyclohexane-1-carboxylate (SMCC) in dimethyl sulfoxide (DMSO).</li>
		<li>Prepare an SPDP solution consisting of 20 mg/mL of succinimidyl 3-(2-pyridyldithio) propionate (SPDP) in DMSO.</li>
		<li>Prepare 20 mg/mL LC-LC-biotin in DMSO.</li>
		<li>Prepare 10 mg/mL horseradish peroxidase (HRP) in phosphate buffered saline (PBS).</li>
		<li>Prepare 10 mg/mL streptavidin (SA) in PBS.</li>
		<li>Prepare 10 mg/mL bovine serum albumin (BSA) in PBS.</li>
		<li>Prepare 100 mM dithiothreitol (DTT) in DI water.</li>
		<li>Prepare 5 mM ethylenediaminetetaacetic acid (EDTA) in PBS.</li>
		<li>Prepare 0.5% casein in PBS.</li>
		<li>Prepare 20% Tween 80 stock in DI water.</li>
		<li>Prepare 0.05% Tween 80 (from 20% stock) in PBS.</li>
		<li>Prepare 50 mM of sodium acetate in DI water.</li>
		<li>Prepare 1% TMB in DMSO.</li>
		<li>Prepare 3% H2O2 in DI water.</li>
		<li>Prepare 2 M H2SO4 in DI water.</li>
	</ol>
	</li>
	<li>Add 1.4 µL of the SPDP solution to 20 µL SA solution.</li>
	<li>Incubate the solution from 5.2 for 1.5 h at RT, wrapped in aluminum foil to avoid exposure to light. This step allows the SPDP crosslinker to bond to SA via an amino group forming a pyridyldithio-activated SA.</li>
	<li>Add 2.4 µL of the DDT solution to the solution from step 5.3 and incubate for 1 h at RT. This allows for a pyridine 2-thione cleavage, resulting in a sulfhydryl-activated SA.</li>
	<li>Add 7.5 µL SMCC solution to 72 µL HRP solution and incubate for 1.5 h at RT, wrapped in aluminum foil to avoid exposure to light. This results in a maleimide-activated HRP bound by an amino group.</li>
	<li>Mix 17 µL LC-LC-biotin solution with 200 µL BSA solution and incubate for 1.5 h at RT, wrapped in aluminum foil to avoid exposure to light. This step allows biotin to conjugate to BSA via an amide bond.</li>
	<li>Transfer the solutions from steps 5.4, 5.5 and 5.6 to separate centrifugal concentrators within spin tubes.</li>
	<li>For tubes containing SA-SPDP, from step 5.4, centrifuge at 10,000 x g until the tube volume reaches 100 µL or for 16 min. Fill the spin tube to 500 µL with PBS-EDTA.
	<ol>
		<li>Repeat step 5.8 five times. Store the stock at 4 °C.</li>
	</ol>
	</li>
	<li>For tubes containing HRP-SMCC, from step 5.5, centrifuge at 10,000 x g until the volume reaches 25 µL or for 16 min. Fill the spin tube to 500 µL with PBS.
	<ol>
		<li>Repeat step 5.9 five times. Store the stock at 4 °C.</li>
	</ol>
	</li>
	<li>For tubes containing BSA-biotin, from step 5.6, centrifuge at 10,000 x g until the volume reaches 100 µL or for 12 min. Fill the spin tube to 500 µL with PBS.
	<ol>
		<li>Repeat step 5.10 four times.</li>
		<li>Centrifuge at 10,000 x g until the volume reaches 100 µL or for 12 min. Add 100 µL of PBS to the tube. Store the stock at 4 °C.</li>
	</ol>
	</li>
	<li>Add 25 µL of the 10 mg/mL HRP solution with 25 µL of 10 mg/mL SA solution and store at 4 °C overnight. This causes the SA to become conjugated with the HRP via a thioether bond.
	<ol>
		<li>Prepare a 1:4 working solution with the overnight solution and store in 4 °C fridge. Store remaining solution at -20 °C.</li>
	</ol>
	</li>
	<li>Prepare two solutions consisting of BSA-Biotin (or BSA) in PBS, by adding 10 µL of BSA-biotin stock (or 10 µL BSA stock) to 490 µL of PBS.</li>
	<li>Add 100 µL of the solution from step 5.12 to each well of a 96-well polystyrene plate.</li>
	<li>Incubate the plate at 37 °C for 1 h, wrapped in foil.</li>
	<li>Wash the wells of the plate with 0.05% Tween 80.
	<ol>
		<li>Add 200 µL of 0.05% PBS-Tween 80 to the wells. Incubate for 2 min at RT. Decant the liquid.</li>
		<li>Repeat step 5.15.1 three times.</li>
	</ol>
	</li>
	<li>Add 200 µL of the 0.5% casein solution to each of the wells in the 96-well plate.</li>
	<li>Incubate the plate at 37 °C for 1 h.</li>
	<li>Repeat step 5.15 to wash the wells three times.</li>
	<li>Prepare a solution by mixing 12 mL of 0.5% casein solution with 7.5 µL of 0.05% Tween 80.</li>
	<li>Dilute the SA-HRP stock 1:10,000 using the solution prepared in step 5.19.</li>
	<li>Add 80 µL of the solution prepared in step 5.20 to each well of a 96-well plate.</li>
	<li>Add 20 µL of the prepared biotin sample to each well of the polystyrene plate. The supernatant prepared in 4.6 can be used as the biotin sample in this step.</li>
	<li>Incubate the plate at 37 °C for 1 h. This step allows for competitive binding between free biotin and immobilize BSA-biotin for SA-HRP binding sites.</li>
	<li>Repeat step 5.15 to wash the wells three times.</li>
	<li>Mix 50 mM sodium acetate solution, 1 % TMB solution, and 3% H2O2 solution at a 1,000:10:1 ratio (20 mL total volume).</li>
	<li>Add 200 µL of the solution from step 5.25 to each well and allow to sit for 15 min at RT, covered with foil.</li>
	<li>Add 50 µL of 2 M H2SO4 to each well to stop the reaction. Measure OD450 using a plate reader.</li>
</ol>
6. Direct Control Scheme Functionalized Surface Preparation
<ol>
	<li>Prepare the following solutions.
	<ol>
		<li>Prepare 20 mg/mL LC-LC-biotin in DMSO.</li>
		<li>Prepare 10 mg/mL HRP in PBS.</li>
		<li>Prepare 0.17 µg/mL SA in PBS.</li>
		<li>Prepare 0.5% casein in PBS.</li>
		<li>Prepare 20% Tween 80 stock in DI water.</li>
		<li>Prepare 0.05% Tween 80 (from 20% stock) in PBS.</li>
		<li>Prepare 50 mM of sodium acetate in DI water.</li>
		<li>Prepare 1% TMB in DMSO.</li>
		<li>Prepare 3% H2O2 in DI water.</li>
		<li>Prepare 2 M H2SO4 in DI water.</li>
	</ol>
	</li>
	<li>Add 7.5 µL LC-LC-biotin to 72 µL HRP and incubate at for 1.5 h at RT, wrapped in aluminum foil to avoid exposure to light. This step causes biotin to conjugate to HRP via an amide bond.</li>
	<li>Transfer the solution from step 6.2 to a centrifugal concentrator within a spin tube
	<ol>
		<li>Centrifuge the solution at 10,000 x g until the volume reaches 100 µL or for 12 min. Fill the spin tube to 500 µL with PBS and repeat the centrifugation and PBS addition four times. Store the stock at 4 °C.</li>
	</ol>
	</li>
	<li>Add the 100 µL of the SA solution from 6.1.3 to each well within a 96-well polystyrene plate.</li>
	<li>Incubate the plate at 37 °C for 1 h, wrapped in foil.</li>
	<li>Wash the wells of the plate with 0.05% Tween 80.
	<ol>
		<li>Add 200 µL of 0.05% Tween 80 to the wells. Incubate for 2 min at RT. Decant the liquid.</li>
		<li>Repeat 6.6.1 three times.</li>
	</ol>
	</li>
	<li>Add 200 µL of the 0.5% casein solution to each of the wells in the 96-well plate.</li>
	<li>Incubate the plate at 37 °C for 1 h.</li>
	<li>Repeat step 6.6 three times to wash the wells.</li>
	<li>Dilute the biotin-HRP stock 1:10,000 using the solution prepared in step 6.3.</li>
	<li>Add 80 µL of the solution prepared in step 6.10 to each well of a 96-well plate.</li>
	<li>Add 20 µL of the prepared biotin sample to each well of the polystyrene plate. The supernatant prepared in 4.6 can be used as the biotin sample in this step.</li>
	<li>Incubate the plate at 37 °C for 1 h. This step allows for competitive binding between free biotin and biotin-HRP for immobilized SA binding sites.</li>
	<li>Repeat step 6.6 to wash the wells three times.</li>
	<li>Mix 50 mM sodium acetate solution, 1% TMB solution, and 3% H2O2 solution at a 1,000:10:1 ratio (20 mL total volume).</li>
	<li>Add 200 µL of the solution from step 6.15 to each well and incubate for 15 min at RT, covered with foil.</li>
	<li>Add 50 µL of 2 M H2SO4 solution to each well to stop the reaction. Measure OD450 using a plate reader.</li>
</ol>

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<li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complete+Chemical+Synthesis%2C+Assembly%2C+and+Cloning+of+a+Mycoplasma+genitalium+Genome%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome.</a> Science. 319, 1215-1220 (2008).</li>
<li>Diamandis, E. P., Christopoulos, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biotin-%28strept%29avidin+system%3A+principles+and+applications+in+biotechnology%5BTitle%5D)+AND+%22Clin.+Chem%22%5BJournal%5D)">The biotin-(strept)avidin system: principles and applications in biotechnology.</a> Clin. Chem. 37, 625-636 (1991).</li>
<li>Nerurkar, L. S., Namba, M., Brashears, G., Jacob,, Lee, A. J., Sever, Y. J., L, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+detection+of+herpes+simplex+virus+in+clinical+specimens+by+use+of+capture+biotin-streptavidin+enzyme-linked+immunosorbent+assay%5BTitle%5D)+AND+%22J.+Clin.+Micro%22%5BJournal%5D)">Rapid detection of herpes simplex virus in clinical specimens by use of capture biotin-streptavidin enzyme-linked immunosorbent assay.</a> J. Clin. Micro. 20, 109-114 (1984).</li>
<li>Cui, Y., Wei, Q., Park, H., Lieber, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanowire+Nanosensors+for+Highly+Sensitive+and+Selective+Detection+of+Biological+and+Chemical+Species%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Nanowire Nanosensors for Highly Sensitive and Selective Detection of Biological and Chemical Species.</a> Science. 293, 1289-1292 (2001).</li>
</ol>

The authors have nothing to disclose.

We have presented a new strategy for interfacing engineered living cells with a functionalized material surface. This was accomplished by developing a cell line capable of synthesizing elevated levels of biotin when induced with IPTG. The elevated levels of biotin may then be used to modify the functionalized surface. The protocols detailed how to engineer the E. coli cell line and how to create two different functionalized surfaces.
Critical steps in this protocol occur throughout th...

Here, we report the procedures for developing a programmable substrate capable of responding to a chemical signal from an engineered cell line.1 We do this by creating a biotin-streptavidin interface that responds to biotin produced by synthetically engineered Escherichia coli (E. coli) cells. Previously, programmable surfaces have been engineered for a wide range of applications from toxin detection2 and point-of-care diagnosis3 to defense and security.4 While programmable surfaces can be useful as sensors and actuators, they can be made &#34;smarter&#34; by endowing them with the ability to adapt to different environmental challenges. In contrast, even simple microorganisms, such as E. coli, have inherent adaptability and are capable of responding to challenges with sophisticated and often unexpected solutions. This adaptability has enabled E. coli populations, controlled by their complex gene networks, to cost-effectively seek resources,5 create value-added products,6 and even power micro-scale robotics.7 By coupling the adaptive advantages of living cells with the use of programmable surfaces, we can create a smart substrate capable of responding to different environmental conditions.
Synthetic biology has given researchers new abilities to program the behavior of living organisms. By engineering cells to contain new gene regulatory networks, researchers can design cells that exhibit a range of programmed behaviors.8,9 Beyond basic research, these behaviors may be used for applications such as controlling material assembly and biologically producing value-added products.10 Herein, we detail how we used the tools of synthetic biology to engineer an E. coli strain that synthesizes biotin upon induction. This strain was developed by using restriction enzyme cloning methods to assemble a plasmid, pKE1-lacI-bioB. This plasmid, when transformed into E. coli strain K-12 MG1655, endows cells with the ability to express elevated levels of bioB, an essential enzyme for biotin synthesis. When transformed cells were induced with isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) and provided with a biotin precursor, desthiobiotin (DTB), elevated levels of biotin were produced.
Biotin&#39;s binding interaction with streptavidin is one of the strongest non-covalent bonds found in nature. As such, the biotin-streptavidin interaction is both well-characterized and highly employed in biotechnology.11 Within this manuscript, we present two strategies employing the biotin-streptavidin interaction to sense and detect cell-produced biotin with a functionalized surface. We refer to these contrasting surfaces as &#34;indirect&#34; and &#34;direct&#34; control schemes. In the indirect control scheme, cell-produced biotin competes with biotin that has been conjugated and immobilized on a polystyrene surface for streptavidin binding sites. In addition, the streptavidin is conjugated with horseradish peroxidase (HRP). HRP modifies 3, 3&#39;, 5, 5&#39;-tetramethylbenzidine (TMB), to produce an optical signal,12 which may be monitored by quantifying the spectral absorbance (i.e., optical density) at 450 nm (OD450). Thus, the indirect control scheme allows researchers to measure cell-produced biotin by monitoring the attentuation of the OD450 signal.
The direct control scheme exploits the streptavidin-biotin event by immobilizing streptavidin directly to a material surface and allowing cell-produced biotin and biotinylated HRP to compete for streptavidin binding sites. Again, the relative levels of cell-produced biotin are monitored by measuring an OD450 signal.
Taken together, the engineered cells and functionalized surfaces allow us to control the properties of a programmable surface by inducing networks in living cells. In other words, we have created a system that takes advantage of the adaptability of living organisms and the reliability and specification of an engineered material interface by linking these systems together.

<table><tbody><tr><td>LB Broth, Miller&amp;nbsp;</td><td>Fisher Scientific</td><td>12-795-027</td><td></td></tr><tr><td>Agar</td><td>Fisher Scientific</td><td>BP9744500</td><td></td></tr><tr><td>Carbenicillin&amp;nbsp;</td><td>Fisher Scientific</td><td>BP26481</td><td></td></tr><tr><td>M9, Minimimal Salts, 5x</td><td>Sigma-Aldrich</td><td>M6030</td><td></td></tr><tr><td>Casamino Acids&amp;nbsp;</td><td>Fisher Scientific</td><td>BP1424-100</td><td></td></tr><tr><td>Magnesium Sulfate, Anhydrous</td><td>Fisher Scientific</td><td>M65-500</td><td></td></tr><tr><td>Calcium Chloride, Dihydrate</td><td>Fisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>Dextrose (D-Glucose), Anhydrous</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>NEB Turbo Cell Line</td><td>New England Biolabs</td><td>C2984l</td><td></td></tr><tr><td>Oligonucleotide Primers</td><td>Thermo Fisher Scientific</td><td>N/A</td><td>25N synthesis, DSL purification</td></tr><tr><td>Q5 High-Fidelity Polymerase</td><td>New England Biolabs</td><td>M0491S</td><td></td></tr><tr><td>Q5 Reaction Buffer</td><td>New England Biolabs</td><td>B9027S</td><td></td></tr><tr><td>dNTP Solution Mix</td><td>New England Biolabs</td><td>N0447S</td><td></td></tr><tr><td>Agarose</td><td>Bioexpress</td><td>E-3120-125</td><td></td></tr><tr><td>Ethidium Bromide, 1%</td><td>Fisher Scientific</td><td>BP1302-10</td><td></td></tr><tr><td>Gel Extraction Kits</td><td>Epoch Biolabs</td><td>2260250</td><td></td></tr><tr><td>GenCatch Plasmid DNA Miniprep Kit</td><td>Epoch Biolabs</td><td>2160250</td><td></td></tr><tr><td>AatII</td><td>New England Biolabs</td><td>R0117S</td><td></td></tr><tr><td>SacII</td><td>New England Biolabs</td><td>R0157S</td><td></td></tr><tr><td>HindIII-HF</td><td>New England Biolabs</td><td>R3104S</td><td></td></tr><tr><td>EcoRI-HF</td><td>New England Biolabs</td><td>R3101S</td><td></td></tr><tr><td>Cutsmart Buffer</td><td>New England Biolabs</td><td>B7204S</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>T4 DNA Ligase Reaction Buffer</td><td>New England Biolabs</td><td>B0202S</td><td></td></tr><tr><td>ColiRolle Glass Plating Beads&amp;nbsp;</td><td>EMD Millipore</td><td>7101-3</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-1</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>Fisher Scientific</td><td>BP1755-10</td><td></td></tr><tr><td>NHS-Desthiobiotin (DTB)</td><td>Thermo Fisher Scientific</td><td>16129</td><td></td></tr><tr><td>Succinimidyl Trans-4-(maleimidylmethyl) Cyclohexane-1-Carboxylate (SMCC)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>S1534</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)&amp;nbsp;</td><td>Fisher Scientific</td><td>BP231-100</td><td></td></tr><tr><td>Succinimidyl 3-(2-pyridyldithio) Propionate (SPDP)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>S1531</td><td></td></tr><tr><td>NHS-LC-LC-biotin</td><td>Thermo Fisher Scientific</td><td>21343</td><td></td></tr><tr><td>Horseradish Peroxidase (HRP)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>31490</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS), 10x Solution</td><td>Fisher Scientific</td><td>BP399500</td><td></td></tr><tr><td>Streptavidin (SA)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>21145</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Fisher Scientific</td><td>BP1600-100</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Fisher Scientific</td><td>BP172-5</td><td></td></tr><tr><td>Ethylenediaminetetaacetic acid (EDTA)&amp;nbsp;</td><td>Fisher Scientific</td><td>S311-500</td><td></td></tr><tr><td>Tween 80&amp;nbsp;</td><td>Fisher Scientific</td><td>T164-500</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Fisher Scientific</td><td>H325-4</td><td></td></tr><tr><td>3, 3&#039;, 5, 5&#039;-tetramethylbenzidine (TMB)</td><td>Fisher Scientific</td><td>AC229280050</td><td></td></tr><tr><td>Vivaspin 500 Centrifugal Concentrators&amp;nbsp;</td><td>Viva Products</td><td>VS0192</td><td></td></tr><tr><td>Sodium Acetate, Anhydrous</td><td>Fisher Scientific</td><td>BP333-500</td><td></td></tr><tr><td>96-Well Polystyrene Plates</td><td>Thermo Fisher Scientific</td><td>266120</td><td></td></tr></tbody></table>

using synthetic biology to engineer living cells that interface with programmable materials

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

Representative results are presented in the accompanying five figures. First, we present the cloning process graphically (Figure 1) so that the reader can visually follow the critical steps for creating the synthetically engineered E. coli strain. In order to characterize the population dynamics of the cells, we provide a growth curve (Figure 2) generated by measuring the optical density at 600 nm (OD600) of the population. Then, we sh...

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

The overall goal of these procedures is to use synthetically engineered E.coli to control and manipulate programmable material surfaces by combining genetic modification and surface functionalization strategies. This method can help answer key questions in fields such as molecular medicine and environmental monitoring. By using genetically programmed cells to interpret a local environment and modify a functionalized material accordingly, we are providing a modular tool for a wide variety of applications.The main advantage of this technique is that living cells are able to act as dynamic sensors capable of reading, processing and recording the conditions around them via functionalized interfaces. After preparing solutions and collecting the biotin-enriched supernatant from biotin-producing E.coli according to the text protocol add 1.4 microliters of SPDP solution to 20 microliters of streptavidin or SA solution. Wrap the tube in aluminum foil and incubate it at room temperature for one and a half hours to allow the SPDP cross linker to bond to SA via an amino group, forming a pyridyldithio-activated SA.Following the incubation, add 2.4 microliters of DTT solution to the tube and incubate the sample at room temperature for one hour to allow a pyridine-2-thione cleavage resulting in a sulfhydryl-activated SA.Next, add 7.5 microliters of SCC solution to 72 microliters of HRP solution, wrap it in aluminum foil, and incubate it at room temperature for one and a half hours.This results in a maleimide-activated HRP bound by an amino group. Mix 17 microliters of LC-LC biotin solution with 200 microliters of BSA solution, wrap the tube in foil and incubate the sample at room temperature for one and a half hour. This step allows biotin to conjugate to BSA via an amide bond.Transfer the SA-SPDP solution, the HRP-SMCC and the BSA-conjugated biotin solution to separate centrifugal concentrators in spin tubes. Centrifuge the SA-SPDP at 10, 000 times g until the tube volume reaches 100 microliters or for 16 minutes, then fill the spin tube to 500 microliters using PBS-EDTA before repeating the spin and the addition of PBS-EDTA five more times. After spinning the solution down to 100 microliters, store the stock at four degrees Celsius.For the HRP SMCC, centrifuge the tubes at 10, 000 times g until the volume reaches 25 microliters or for 16 minutes. Fill the spin tube to 4500 microliters using PBS before repeating the spin and the addition of PBS five more times. After spinning the solution down to 100 microliters, store the stock at four degrees Celsius.For the BSA biotin, centrifuge the sample at 10, 000 times g until the volume reaches 100 microliters or for 12 minutes, then fill the spin tube to 500 microliters using PBS. After repeating the spin and the addition of PBS four more times, centrifuge the tube until the volume reaches 100 microliters or for 12 minutes, then add 100 microliters of PBS to the tube and store the stock at four degrees Celsius. Next, combine 25 microliters of the 10 milligram per milliliter HRP with 25 microliters of 10 milligram per milliliter SA solution and store the tube at four degrees Celsius overnight, then prepare two aliquots of BSA biotin in PBS by adding 10 microliters of BSA biotin stock to 490 microliters of PBS.Pipette 100 microliters of the solution into each well of a 96 well plate. Wrap the plate in foil and incubate it at 37 degrees Celsius for one hour. Next, add 200 microliters of 05%Tween 80 and BPS to the wells.Incubate the plate at room temperature for two minutes, then aspirate and decant the liquid. After repeating the wash three times, add 200 microliters of the 0.5%casing solution to each of the wells, then incubate the plate at 37 degrees Celsius for an hour. Following another round of washes as before, mix 12 milliliters of 05%casing solution with 7.5 microliters of 05%Tween 80, then use this solution to dilute the SA-HRP stock solution one to 10, 000.Pipette 80 microliters of the diluted SA-HRP into each well of a 96 well plate, then add 20 microliters of the prepared biotin sample to each well of the plate. Incubate the plate at 37 degrees Celsius for one hour. This step allows for competitive binding between free biotin and immobilized BSA biotin for SA-HRP binding sites.Then after washing the plate three times as before, prepare a 20 milliliter solution of 50 millimolar sodium acetate, 1%TMB and 3%hydrogen peroxide at a 1, 000 to 10 to one ratio. Add 200 microliters of the solution to each well of the plate, wrap it in foil and incubate it at room temperature for 15 minutes. Pipette 50 microliters of two molar sulfuric acid into each well to stop the reaction, then use a plate reader to measure the OD 450.Following the preparation of solutions according to the text protocol, add 7.5 microliters of LC-LC biotin to 72 microliters of HRP. Wrap the solution in foil and incubate the sample at room temperature for one and a half hours with agitation. This step causes biotin to conjugate to HRP via an amide bond.Transfer the solution to a centrifugal concentrator within a spin tube and centrifuge the solution at 10, 000 times g until the volume reaches 100 microliters or for 12 minutes, then use PBS to fill the spin tube to 500 microliters and repeat the centrifugation and buffer addition four times. Next, add 100 microliters of 0.17 micrograms per milliliter SA in PBS to each well of a 96 well plate. Wrap the plate in foil and incubate it at 37 degrees Celsius for an hour.To wash the wells of the plate, pipette 200 microliters of 05%Tween 80 into each well. Incubate the plate at room temperature for two minutes and decant the liquid. After repeating the wash three more times, add 200 microliters of the 0.5%casing solution to the wells.Incubate the plate at 37 degrees Celsius for one hour before washing the wells three times as before. Using the washed and concentrated biotin HRP solution previously prepared, dilute the biotin HRP stock solution one to 10, 000, then add 80 microliters of the solution to the wells of a 96 well plate. Add 20 microliters of prepared biotin sample to each well of the plate and incubate the plate at 37 degrees Celsius for one hour to allow competitive binding between free biotin and biotin HRP for the immobilized SA binding sites.After washing the wells three times as before, prepare a 20 milliliter solution of 50 millimolar sodium acetate, 1%TMV, and 3%hydrogen peroxide at a 1, 000 to 10 to one ratio. Add 200 microliters of the solution to each well of the plate, cover it with foil, and incubate it at room temperature for 15 minutes, then add 50 microliters of two molar sulfuric acid to each well to stop the reaction. Finally, use a plate reader to measure the OD 450.The engineered inducible E.coli MG1655 wild type cells were grown and monitored in minimal medium as well as minimal medium supplemented with DTB and/or IPTG. These plots shows the OD 600 reading measured every five minutes for 24 hours. In this experiment, the fluorescent protein mCherry was used in place of the bioB gene so the induction profile could be measured when engineered cells were induced with IPTG.These results demonstrate the efficacy of the inducible gene network. Plotted here are the optical signals at OD 450 in response to varying concentrations of biotin for both the indirect and direct functionalized surface schemes. In this experiment, products of induced engineered cells were used to chemically modify the functionalized surface.The different biotin concentrations are presented as measured by the indirect control scheme functionalized surface for wild type cells, uninduced cells containing the pKE1-lacI-bioB plasmid and induced cells containing the pKE1-lacI-bioB plasmid. Once mastered, cells can be genetically engineered in two days, while the direct and indirect surface functionalization techniques can be each done in five hours if they are performed properly. While attempting this procedure, it's important to remember to avoid contamination of bacterial cell culture and cover the surfaces during functionalization to avoid photo bleaching.After watching this video, you should have a good understanding of how to functionalize surfaces for both the direct and indirect control schemes. Don't forget that working with ethidium bromide can be extremely hazardous, and precautions, such as wearing gloves, should always be taken while performing this procedure.

using-synthetic-biology-to-engineer-living-cells-that-interface-with

Research

JoVE Journal

Bioengineering

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Here, an automated workflow to perform modular DNA "device" assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

Rapid Characterization of Genetic Parts with Cell-Free Systems

This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell development.

Cell-Free Protein Synthesis System for Building Synthetic Cells

Here, a protocol for designing, constructing, and delivering tetracycline-controlled based inducible synthetic circuit is presented. The effect of the synthetic circuits on parasite elimination and cytokine expression can be studied by channelizing the presented pipeline. This protocol is relevant for researchers studying synthetic circuit-based therapeutics in infectious diseases.

Immunometabolic Circuits in Infection for Advancing Host Directed Therapies

This article describes a biofabrication approach:  deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Bridging the Bio-Electronic Interface with Biofabrication

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Learn to engineer E. coli for controlling programmable material surfaces using genetic modification and surface functionalization. This method enables dynamic sensing for molecular medicine and environmental monitoring applications.

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

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