Name: using synthetic biology to engineer living cells that interface with programmable materials
Uploaded: 2017-03-09T15:00:00
Description: Watch this Scientific Journal Video about Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials at JoVE.com

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 &#176;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 ...

<ol>
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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.

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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...

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

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.

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