구현을위한 프로토콜

The overall goal of this procedure is to create an e coli based cell-free expression system referred to as transcription translation or TX tl. This is accomplished by first making three initial components for TX TL crude cell extract, amino acid solution and energy solution. The amino acid solution and energy solution will be later combined to make the TX TL buffer.

The second step is to calibrate the crude cell extract to determine optimal magnesium, potassium, and DTT concentrations to produce TX TL reactions with maximum levels of expression. Next, the calibration results are used to make a three tube TX TL system made of buffer crude cell extract and DNA. The final step is to execute a TX TL reaction using the reagents just made.

Ultimately, TX TL is used to demonstrate synthetic biology circuits as well as traditional cell-free expression applications. This method can help test circuits in the field of synthetic biology by providing an in vitro equal environment, which emulates that in vivo. The implications of this technique extend towards increasing the speed of synthetic biological design by removing the need to conduct all prototyping steps in vivo.

Assisting a procedure will be Claire Hayes, a research assistant in our group. Please note that this video will go through only select sections of the protocol critical for filming. The full protocol is available with the text article.

Earlier in this protocol, bacterial cells were grown and pelleted. Now the bacterial cells will be lysed using a bead beater. Keep all 50 milliliter falcon tubes containing cell suspension on ice.

For the purposes of this video, bead beading will be demonstrated for only one tube. The beads must be added intermittently to the Falcon tube in three aliquots each using one third of the total beads. Add the first aliquot of beads to the tube vortex for 30 seconds and place on ice.

In the same way, add the second aliquot of beads, vortex, and place on ice. After adding the last aliquot and vortexing ensure the beads are uniformly distributed, a thick paste should be formed after the third vortexing step. Place the tube on ice.

Prepare a five milliliter volume pipette tip by using a sterile pair of scissors to cut off the end. To create a three to four millimeter opening, dial the pipette to two milliliters. Place 20 sterile bead beading tubes on ice.

Use the modified pipette to verify the high viscosity of the bead cell solution. It should be viscous to the point of barely exiting the pipette tip. During ejection, remove the bead cell solution from the Falcon tube and transfer into a bead beading tube.

Filling it three quarters full spin extremely briefly in a mini centrifuge. To remove air bubbles without redistributing the beads, finish adding bead cell solution to the tube to form a concave meniscus. Next, add a very small drop of bead cell solution onto the inside of a bead beading tube cap.

Being careful to not put solution into the outside lip of the cap. Otherwise, the bead beading tube will not close Sufficiently. Tap the cap on a flat surface and verify that there are no air bubbles on the bottom of the cap.

Cap the bead beating tube. If done correctly, the cap should be tightly sealed. No air bubbles should be visible and little if any bead cell solution should overflow from here on, two people are required to accomplish the bead beading efficiently.

Hand the filled tube to an assistant for bead beading while the first demonstrator continues to fill more bead beading tubes with the remaining bead cell solution. Take the filled bead beading tube and place it on ice. Once two filled bead beading tubes have been collected and have been on ice for at least a minute.

Begin bead beading bead one tube for 30 seconds at 46 RPM. Place upside down on ice for 30 seconds while beating the other tube. Repeat the beading and icing such that each filled bead beading tube is beaten for a total of one minute.

Once all filled bead beading tubes have been processed, construct a filter apparatus from a 15 milliliter falcon tube. First, add a new bead beading cap flat part face up to the bottom of the tube. Then remove the cap from a processed bead beading tube and press a micro chromatography column firmly onto the end of the processed bead beading tube until completely sealed.

Snap off the elucian end of the micro chromatography column and place it elucian. End down into an empty bead beading tube. Place this complex into the 15 milliliter falcon tube.

Construct the filter apparatuses for all filled bead beating tubes and keep them on ice. When complete centrifuge, the filter apparatuses Falcon tube uncapped at 6, 000 G for five minutes at four degrees Celsius to separate extract and pellet from beads. After centrifugation, verify that each bead beading tube has produced viable extract.

Properly beaten extract will not be turbid and the pellet will have two distinct layers as illustrated by the tube on the left. Turbid tubes as shown in the example on the right must be discarded. Transfer the supernatant from non turid tubes into individual 1.75 milliliter micro centrifuge tubes, taking as little pellet as possible.

Keep on ice until all bead beading tubes have been processed. Next, spin down the micro centrifuge tubes and collect the supernatant. After consolidating 500 microliters of pellet free supernatant into a new bead beading tube.

Incubate the tubes with caps removed at 220 RPM and 37 degrees Celsius for 80 minutes. This will digest remaining nucleic acids using endogenous exonuclease released during the bead beading process. When the incubation is complete, the extract should look turbid.

Consolidate extract in 1.75 milliliter micro centrifuge tubes up to 1.5 milliliters per tube centrifuge at 12, 000 G for 10 minutes at four degrees Celsius. Using a pipette consolidate pellet free supernatant into either a 1.75 milliliter micro centrifuge tube for smaller yields or a 15 milliliter falcon tube for larger yields on ice. Cap the tube and mix well by inverting.

Save 10 microliters of supernatant on ice for later measurement of protein concentration. Determine total amount of extract produced and hydrate the necessary number of 10 molecular weight cutoff dialysis cassettes by submersing in S 30 B buffer for two minutes. Load cassettes with up to 2.5 milliliters of extract.

Each beaker can take up to two cassettes dialyze stirring at four degrees Celsius for three hours. Refer to the written article for processing steps after dialysis. A basic transcription translation reaction has three parts, crude cell extract, buffer and DNA.

Although reactions can vary in volume, this protocol utilizes a pre-written template to conduct a 10 microliter reaction.Here. Items in purple indicate user input values, and items in blue indicate additional reagents to add to the reaction design the experiment in sili using the master mix preparation section and DNA preparation section. Generally constants can be put into the master mix preparation section while variables can be put into the DNA preparation section.

Minimize samples per experiment to avoid sample evaporation and experimental start time bias. A sample setup is shown in this table. To prepare DNA samples for each sample, ID aliquot out the indicated DNA water and user supplied items into a micro centrifuge tube.

At room temperature, prepare the master mix consisting of buffer extract and any global user supplied items keeping on ice and vortexing. After the addition of each item, add the appropriate amount of master mix to each DNA sample and keep at room temperature. Treat this as the reaction start time.

Vortex each sample and centrifuge at 10, 000 G for 30 seconds at room temperature to bring down any residual sample and to reduce bubbles. Run the reaction in a 384 well plate at 29 degrees Celsius. Run times will vary depending on the experiment, but typically last under eight hours.

At the completion of the run, the data can be read from a plate reader. This paper presents a five day protocol for the preparation of an endogenous e coli based transcription translation or TXTL cell-free expression system. The expression conditions of this system were optimized through testing the effects of different plasmid processing methods and the elucian buffer.

Certain variables introduced into the TXTL system should be calibrated beforehand for toxicity, for example, in the comparison of plasmid processing methods purification method one uses only a kaya prep spin mini prep kit while in purification method two, the plasmid is prepared using the same mini prep kit and then post-process with a kayak. Quick PCR purification kit. This graph shows endpoint fluorescence after eight hours as well as maximal rate of protein production based on a 12 minute moving Average error bars are one standard deviation from four independent runs on different days.

The difference in expression observed is due to the difference in salt content. However, other items may show no effect on TXTL such as elucian buffer. Different concentrations of TS chloride were compared in a cell-free expression reaction based on the expression of one ano molar of plasmid.

Concentrations given are final concentrations of tris chloride. In the reaction elucian buffer used is 10 millimolar tris chloride error bars are one standard deviation from three independent runs on different days. This figure shows typical calibration plots for crude cell extract, calibrated for additional magnesium glutamate, potassium glutamate, and DTT levels.

Endpoint fluorescence after eight hours as well as maximal rate of protein production based on a 12 minute moving average are shown. Note that every crude cell extract needs to be calibrated independently for these three variables. In general, the results indicate that the crude cell extract is most sensitive to magnesium glutamate levels followed by potassium glutamate levels.

Based on these plots, an acceptable range of additional magnesium glutamate is four millimolar potassium glutamate is 60 to 80 millimolar and DTT is zero to three millimolar airing on the lower side. The end efficiency of each crude cell extract preparation can vary based on user proficiency and on environmental conditions. Although typical yield variation is between five and 10%endpoint fluorescence of two crude extracts prepared on different dates is shown here.

Error bars are one standard deviation from three independent runs on different days. To demonstrate the cell-free expression system, a negative feedback loop based on Tet repression was constructed and tested the same circuit run with and without a TC showed a sevenfold endpoint expression. Change of D-E-G-F-P reporter after eight hours of expression error bars are one standard deviation from three independent runs on different days.

The genetic circuit is shown in the inset. This final figure depicts the cost and expression analysis of competing crude cell extracts. The pie chart in a breaks down the costs of labor and materials of the TX TL cell-free expression system based on costs of reagents as of December, 2012 and labor costs of 14 per hour.

Panel B compares the TX cell-free expression system costs versus other commercial systems. Costs are broken down per microliter, although reaction volumes may vary per kit. Material costs for this system are about 3 cents per microliter reaction, which is a 98%cost reduction compared to comparable commercial cell-free systems.

Panel C shows a comparison of the TX TL cell-free expression system yield versus other commercial systems. This system can produce up to 0.75 milligrams per milliliter of reporter protein using either a Sigma 70 based promoter with Lambda phage operators or a T seven driven promoter. These comparisons indicate that the TX TL cell-free expression system can produce equivalent amounts of protein as T seven based systems add a tremendous cost reduction to similar commercial systems.

We hope this technique paves the way for simplifying engineering process in synthetic biology.