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.
