The authors acknowledge the Armenise-Harvard Foundation, the Marie-Curie Trentino COFUND (ACS), the Autonomous Province of Trento (Ecomm), and CIBIO for funding.

1. Preparing the DNA Template
<ol>
	<li>Purify plasmid from a standard laboratory strain of E. coli, such as E. coli DH5a or Nova Blue with a commercial kit. Alternatively, a linear PCR product can be similarly purified with a commercial kit. Elute the DNA with H2O only.</li>
	<li>Phenol-chloroform extract the DNA solution26.</li>
	<li>Determine the DNA concentration and purity, e.g. by UV absorbance or other suitable methods. It is important to use highly pure DNA for efficient transcription-translation.</li>
</ol>
2. Preparing the Thin Lipid Film
<ol>
	<li>Weigh the dry lipid powder and dissolve in solvent. For 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), stock solutions are prepared in chloroform at a concentration of 40 mg/ml. 
	Note: Organic solvents must always be handled with glass pipettes and bottles. In other words, plastic must never be used. Store stock solutions in air tight, solvent resistant amber glass bottles at -20 &#176;C, preferably under argon.</li>
	<li>Aliquot 12 &#956;mol POPC (220 &#956;l&#160;of 40 mg/ml&#160;stock solution) into a 5 ml&#160;round bottom flask.</li>
	<li>Evaporate the solvent with a rotary evaporator (Figure 1) to generate the thin lipid film.
	<ol>
		<li>Securely attach the round bottom flask to the distillation tube with a circular clip and start the rotation of the flask.</li>
		<li>Initiate the circulation of water through the condenser coil. Using a heat bath is not necessary, because chloroform has a low boiling point of 61.2 &#176;C at normal atmospheric pressure.</li>
		<li>Ensure that the system is open to the atmosphere by checking that the stopcock is open. Turn on the vacuum pump and slowly close the stopcock to apply the vacuum to the system.</li>
		<li>A thin lipid film is deposited on the walls of the round bottom flask during rotary evaporation forming an opaque film that is visible by eye. Let the rotary evaporation continue for 0.5-2 hr. To stop the process, slowly release the vacuum pressure, stop the rotation, and remove the round bottom flask.</li>
	</ol>
	</li>
</ol>
3. Lipid Resuspension and Vesicle Homogenization
<ol>
	<li>Add 1 ml&#160;18.2 M&#937; H2O to the thin lipid film directly in the round bottom flask. Vigorously vortex the solution at maximum speed, approximately 3,200 rpm, until the lipid film detaches from the glass, which is observable by eye.</li>
	<li>Transfer the lipid dispersion into a 2 ml&#160;microcentrifuge tube.</li>
	<li>Set-up a ring stand to hold a homogenizer with a 5 mm tip. Place a microcentrifuge stand below the homogenizer to securely hold the lipid dispersion. Rinse the dispersing element of the homogenizer by immersing the tip in 18.2 M&#937; H2O and running for a few seconds.</li>
	<li>Place the dispersing element directly into the lipid dispersion ensuring that the tip does not touch the bottom of the microcentrifuge tube. Homogenize for 1 min at power level 4 or 14,000 rpm.</li>
</ol>
4. Vesicle Extrusion and Lyophilization
<ol>
	<li>Assemble the parts of the mini-extruder (Figure 2), which consists of an outer casing and a retainer nut that houses two Teflon internal membrane supports, two O-rings, and one Teflon bearing.
	<ol>
		<li>Place the two O-rings into the grooves of the internal membrane supports. Prewet two filters and one 400 nm membrane. The filters will be placed against the Teflon inside of the O-rings with the membrane between them.</li>
		<li>Place the membrane supports into the extruder outer casing with the membrane surrounded by two filters in between the O-rings. Place the Teflon bearing inside the casing and attach the retainer nut and tighten by hand.</li>
	</ol>
	</li>
	<li>Rinse two syringes and fill one with 18.2 M&#937; water. Insert the syringe needles into the small holes in the Teflon on either end of the extruder assembly. The needles should slide in easily; do not force the needles. Secure the extruder with the syringes into the extruder housing and fasten.</li>
	<li>Pass the water through the extruder by slowly pushing the water out of one syringe and into the other. This represents one passage. Repeat for a total of three passages ensuring that there are no leaks present. Remove the syringe of water and dispose of the water.</li>
	<li>Fill one syringe with the sample, attach to the extruder, and slowly pass the vesicle solution through the membrane as described above in step 4.3. Repeat 10x&#160;for a total of 11 passages. As the extrusion process proceeds, the sample will become less turbid and easier to push across the membrane. A sudden decrease in resistance, however, usually indicates a rupturing of the membrane.</li>
	<li>Transfer the extruded vesicle solution and make 40 &#956;l&#160;aliquots of the vesicles into microcentrifuge tubes. Flash freeze each aliquot in either dry ice or in liquid nitrogen.</li>
	<li>Lyophilize each aliquot with a centrifugal evaporator overnight at 30 &#176;C. Store the lyophilized empty vesicles at -20 &#176;C.</li>
</ol>
5. Encapsulating Transcription-translation Machinery
<ol>
	<li>Mix the components of the transcription-translation reaction and add 20 units of RNase inhibitor. Incubate on ice.</li>
	<li>Add the DNA template. For a control reaction, 250 ng of a plasmid encoding mVenus or a similar fluorescent protein behind a T7 transcriptional promoter and a strong E. coli ribosome binding site is recommended.</li>
	<li>Bring the final volume to 25 &#956;l&#160;with RNase-free water.</li>
	<li>Hydrate an aliquot of lyophilized vesicles (from step 4.6) with 10 &#956;l&#160;of the reaction assembled in step 5.3. Briefly vortex the mixture until the vesicles are resuspended. This should take less than 30 sec.</li>
	<li>Incubate the reaction on ice for 30 min to allow the vesicles to swell.</li>
	<li>Dilute the vesicle mixture 20-fold to a final volume of 30 &#956;l&#160;by adding 1.5 &#956;l&#160;of vesicles into 27.0 &#956;l&#160;of 50 mM Tris-HCl, 50 mM NaCl, pH 7.4 and 1.5 &#956;l&#160;of 20.2 mg/ml&#160;Proteinase K. DNase and RNase can also be added at this point as an alternative to proteinase K to degrade extravesicular material.</li>
	<li>Incubate for at least 2.5 hr at 37 &#176;C.</li>
</ol>
6. Microscopy
<ol>
	<li>Examine the vesicles and the progress of fluorescent protein production at different time points. The vesicles will have a larger diameter than the 400 nm pore-size of the membrane used for vesicle extrusion.
	<ol>
		<li>Prepare a sample chamber by placing a 20 x 5 mm silicon spacer onto a standard microscope slide. Pipette 10 &#956;l&#160;of vesicles into the sample chamber. Place a siliconized glass cover slip over the chamber.</li>
		<li>Observe the vesicles with a 63X oil-dispersion or similar objective by bright field and fluorescence microscopy using the appropriate filter set for the exploited fluorescent protein.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Although cell-free synthetic biology is still in its infancy, advances have laid a foundation from which increasingly complex cell-like systems can be made. The reconstitution of transcription-translation machinery from fully defined components9&#160;inside of vesicles28&#160;was particularly significant in facilitating later efforts in constructing environmentally responsive artificial cells17,18.&#160;Similarly, artificial cell studies have been used to probe evolutionary processes4,29,30, the mechanistic details of RNA and protein synthesis22,31, the influences of metabolic load32,33,&#160;and the assembly of viral particles34. Importantly, enough knowledge now exists that basic cellular function can be reconstituted inside of vesicles in the laboratory following these previous reports and the protocols described herein.
In addition to being easy, the described encapsulation procedure has several benefits. For example, many empty, lyophilized vesicle aliquots can be made in advance and stored at -20 &#176;C for later use. The protocol does not subject biological molecules to organic solvents, drastic temperature changes, or long periods of dialysis. We expect that the gentleness of the procedure will facilitate the incorporation of additional components as needed. We also have not observed adverse effects to changing the lipid composition of the membrane on encapsulation or transcription-translation efficiency. Therefore, lipids more amenable to the incorporation of membrane proteins, specific morphologies, or visualization could conceivably be exploited.
The major limitation of the described method is that the resulting vesicles are not homogenous in size or lamellarity. For many applications, these difficulties do not interfere with the interpretation of data. However, if needed, additional steps can be incorporated to narrow the size distribution and decrease the layers of membranes, such as further rounds of extrusion after encapsulation, freeze-thawing, or dialysis35.&#160;Undoubtedly better methods that circumvent these and other problems will be developed. Until then, we find the protocol described here to be well suited for the construction of cellular mimics.

Cell-free, in vitro transcription-translation reactions and the generation of vesicles from synthetic lipids are nothing new. However, combining the two into a cellular mimic is significantly more challenging1-6.&#160;E. coli cell extracts with or without T7 RNA polymerase can be used as a source of transcription-translation machinery7,8. Cell extracts benefit from the presence of additional cellular components that can facilitate protein expression and folding. Alternatively, a mix of individually purified RNA and protein molecules, i.e. the PURE system9, can be used to mediate intravesicular protein synthesis4,10-14. The PURE system allows for the construction of fully defined cellular mimics and does not suffer from the nuclease activity found in cell extracts. Practically, this means that much less DNA template is required, thereby facilitating processes with low encapsulation efficiency11. Although less frequently used, cellular mimics can be built with cell extracts derived from eukaryotic cells15.&#160;Thus far, genetically encoded encapsulated cascades and cellular mimics that sense the environment have been reported16-18.
The simplest way to monitor transcription-translation reactions is to measure the fluorescence or luminescence of genetically encoded elements. Typically, firefly luciferase19&#160;or GFP are used, although in vitro reactions are frequently measured by radiolabeling. Fluorescence detection additionally allows for the monitoring of populations of vesicles20,21 through cytometry based methods, thereby offering some insight into the stochastic nature of biological-like processes. These monitoring methods have been used to define a small set of design rules and a library of parts from which to build from, including a collection of fluorescent proteins that are compatible with in vitro transcription-translation22,&#160;the influence of genetic organization on expression22,&#160;the activity of sigma factors16, and the efficiency of transcriptional terminators23. Nevertheless, there remains much that needs to be done to increase the capability of building predictable in vitro, genetically encoded devices.
There are many methods available to make vesicles. The most common methods depend upon the generation of a thin lipid film on a glass surface followed by resuspension in aqueous solution24.&#160;If the aqueous solution contains transcription-translation machinery, for example, then a fraction of the vesicles formed would contain the necessary components for protein production. However, the encapsulation efficiency of such methods is low, meaning that only a small percentage of vesicles are active. Many of the alternative methods characterized by much higher encapsulation efficiency exploit the conversion of water-in-oil emulsion droplets to vesicles. While it is likely that such methods will be commonplace in the future, currently these methods suffer from the need of specialized equipment and give vesicles with altered membrane compositions25. A distinct advantage of water-in-oil to vesicle methods is the potential to control membrane lamellarity. The method described herein is based on the thin lipid film protocol described by the Yomo laboratory11&#160;with slight modifications including an additional homogenization step. This method is easy, cheap, and gives robust vesicles well suited for the encapsulation of transcription-translation machinery.

<table border="0" cellpadding="0" cellspacing="0" width="745">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Quick Spin Mini-prep kit</td>
			<td style="width:243px;">Qiagen</td>
			<td style="width:119px;">27104</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Spectrometer</td>
			<td style="width:243px;">NanoDrop 1000</td>
			<td style="width:119px;">NDB767ND</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">POPC</td>
			<td>Avanti Polar Lipids</td>
			<td style="width:119px;">770557</td>
			<td style="width:167px;">MW 760 g/mol 
			Transition Temp -2 &#176;C 
			CAS# 26853-31-6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">459836</td>
			<td>Anhydrous, &#62;99.5%</td>
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			<td height="63" style="height:63px;width:216px;">Phenol-choloroform-isoamyl alcohol 25:24:1, for molecular biology use</td>
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			<td>Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA</td>
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			<td height="21" style="height:21px;width:216px;">Chloroform</td>
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			<td height="21" style="height:21px;width:216px;">Brown amber glass bottles</td>
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			<td>Buchi Rotovapor R-210/Sigma</td>
			<td>Z563846EU-1EA</td>
			<td>With jack and water bath, 29/32 joint 240 V</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Analog vortex mixer</td>
			<td style="width:243px;">VWR</td>
			<td style="width:119px;">945300</td>
			<td>Speed 1,000-3,200 rpm</td>
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			<td height="21" style="height:21px;">Homogenizer</td>
			<td>IKA T10 Basic Ultra-Turbax</td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-extruder</td>
			<td>Avanti Polar Lipids</td>
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			<td height="21" style="height:21px;width:216px;">Extruder filters</td>
			<td>Whatman</td>
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			<td>drain disc 10 mm&#160;</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Extruder polycarbonate membrane 400 nm</td>
			<td>Whatman</td>
			<td style="width:119px;">61007</td>
			<td>nuclepore polycarbonate&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Speed vacuum</td>
			<td>Labconco</td>
			<td style="width:119px;">7970011</td>
			<td>Centritrap DNA concentrator</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PURExpress kit</td>
			<td style="width:243px;">New England Biolabs</td>
			<td style="width:119px;">NRM #E6800S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAse inhibitor (40,000 U/ml)</td>
			<td style="width:243px;">New England Biolabs</td>
			<td style="width:119px;">#M0307S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K (20.2 mg/ml)</td>
			<td style="width:243px;">Fermentas</td>
			<td style="width:119px;">#EO0491</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:243px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope</td>
			<td height="42" style="height:42px;width:243px;">Zeiss Observer Z1with a AxioCam MRm camera</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RealTime</td>
			<td style="width:243px;">CFX96 Real time PCR Detection System (Biorad)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silicon press to seal&#160;&#160;-Molecular Probe</td>
			<td style="width:243px;">Life Technologies</td>
			<td style="width:119px;">P18174</td>
			<td>Resistant from&#160; -25-30 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Siliconized glass circle cover slides</td>
			<td style="width:243px;">Hampton Research</td>
			<td style="width:119px;">HR3-231</td>
			<td>&#160;&#160; Diameter= 22 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ImageJ</td>
			<td style="width:243px;">NIH&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the encapsulation of cell-free transcription and translation machinery in vesicles for the construction of cellular mimics

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up cellular mimics that better represent the complexity of cellular life. To date there are a number of paths that could be taken to build compartmentalized cellular mimics, including the exploitation of water-in-oil emulsions, microfluidic devices, and vesicles. Each of the available options has specific advantages and disadvantages. For example, water-in-oil emulsions give high encapsulation efficiency but do not mimic well the permeability barrier of living cells. The primary advantage of the methods described herein is that they are all easy and cheap to implement. Transcription-translation machinery is encapsulated inside of phospholipid vesicles through a process that exploits common instrumentation, such as a centrifugal evaporator and an extruder. Reactions are monitored by fluorescence spectroscopy. The protocols can be adapted for recombinant protein expression, the construction of cellular mimics, the exploration of the minimum requirements for cellular life, or the assembly of genetic circuitry.

Fluorescence microscopy reveals that fluorescence is only observed inside of the vesicles, because extravesicular material is enzymatically degraded (Figure 3). For the expression of mVenus, intravesicular fluorescence begins to be observed after 1.5 hr at 37 &#176;C and reaches maximum fluorescence intensity within 6 hr. The optimal temperature and incubation time can vary depending upon the specific constructs used. For example, different fluorescent proteins mature quite differently in a temperature dependent manner. In other words, the observation of protein production is not solely dependent upon protein synthesis and folding but also on chromophore formation. Overall protein synthesis can be increased by incorporating membrane protein pores to allow for an influx of depleted&#160;components necessary for transcription and translation27.
It is recommended to carry out an analogous transcription-translation reaction in the absence of vesicles to ensure that the exploited genetic construct is functional. This control reaction is more easily monitored by fluorescence spectroscopy rather than microscopy.&#160;Figure 4 shows an in vitro transcription-translation reaction of a construct encoding mVenus. Unencapsulated reactions give much higher total fluorescence intensities than similar intravesicular reactions. This is due to encapsulation efficiency and because the total intravesicular volume is much less than the extravesicular volume (i.e. a dilution effect).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51304/51304fig1.jpg" width="600px" /> 
Figure 1. The rotary evaporator and vacuum pump. <a href="https://www.jove.com/files/ftp_upload/51304/51304fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51304/51304fig2.jpg" width="600px" /> 
Figure 2. The housing and parts of the extruder are shown separately. From left to right, syringe, retainer nut, teflon bearing, internal membrane supports with black O-rings facing one another, extruder outer casing, and second syringe. <a href="https://www.jove.com/files/ftp_upload/51304/51304fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51304/51304fig3.jpg" width="600px" /> 
Figure 3. Fluorescence images of mVenus protein production in liposomes.&#160;A&#160;and&#160;C:&#160;Bright field images of multilamellar vesicles after 1.5 hr and 2.5 hr.&#160;B and&#160;D:&#160;The production of mVenus is visualized by fluorescence (colored green) after 1.5 and 2.5 hr, respectively. Scale bar is 20 &#956;m. <a href="https://www.jove.com/files/ftp_upload/51304/51304fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51304/51304fig4.jpg" width="600px" /> 
Figure 4. In vitro control reaction of nonencapsulated in vitro transcription and translation of mVenus. Fluorescence intensity was measured every 5 min over 4 hr. The data were acquired with a Real-Time PCR instrument. <a href="https://www.jove.com/files/ftp_upload/51304/51304fig4highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics at JoVE.com

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

Herein we describe simple methods for the preparation of vesicles, the encapsulation of transcription and translation machinery, and the monitoring of protein production. The resulting cell-free systems can be used as a starting point from which to build increasingly complex cellular mimics.

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

the-encapsulation-cell-free-transcription-translation-machinery

Research

JoVE Journal

Bioengineering

15.0K 조회수.  University of Trento.  여기에서 우리는 소포, 전사 및 번역 기계 캡슐, 단백질 생산의 모니터링 준비를위한 간단한 방법을 설명합니다. 그 결과 세포가없는 시스템은 점점 더 복잡 해지는 세포의 모방을 구축하기 시작 지점으로 사용할 수 있습니다. 

비디오: The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in transcription of non-coding RNAs, specifically of microRNAs. Here, we propose a strategy to study the potential role of transcription factor in regulating transcription of microRNAs using publically available data, computational resources and high throughput data. We use the H3K4me3 epigenetic signature to identify microRNA promoters and chromatin immunoprecipitation (ChIP)-sequencing data from the ENCODE project to identify microRNA promoters that are enriched with transcription factor binding sites. By transfecting cells of interest with shRNA targeting a transcription factor of interest and subjecting the cells to microRNA array, we study the effect of this transcription factor on the microRNA transcriptome. As an illustrative example we use our study on the effect of STAT3 on the microRNA transcriptome of chronic lymphocytic leukemia (CLL) cells.

Herein we propose a strategy to study the effect of a transcription factor of interest on the microRNA transcriptome using publically available data, computational resources and high throughput data from microRNA arrays after transfecting cells with small hairpin (sh)RNA targeting a transcription factor of interest.

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

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

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

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.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous ...

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a natural producer of chloramphenicol, jadomycin, and pikromycin, which makes it an attractive candidate&#160;as a next-generation synthetic biology chassis. Therefore, genetic tools that accelerate the development of S. venezuelae ATCC 10712, as well as other Streptomyces spp. models, are highly desirable for natural product engineering and discovery. To this end, a dedicated S. venezuelae ATCC 10712 cell-free system is provided in this protocol to enable high-yield heterologous expression of high G+C (%) genes. This protocol is suitable for small-scale (10-100 &#956;L) batch reactions in either 96-well or 384-well plate format, while reactions are potentially scalable. The cell-free system is robust and can achieve high yields (~5-10 &#956;M) for a range of recombinant proteins in a minimal setup. This work also incorporates a broad plasmid toolset for real-time measurement of mRNA and protein synthesis, as well as in-gel fluorescence staining of tagged proteins. This protocol can also be integrated with high-throughput&#160;gene expression characterization workflows or the study of enzyme pathways from high G+C (%) genes present in Actinomycetes genomes.

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

This protocol details an enhanced method for synthesizing high yields of recombinant proteins from a Streptomyces venezuelae cell-free transcription-translation (TX-TL) system.

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a ...

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular processes in vitro. In recent years, encapsulation within GUVs has proven to be a helpful approach for reconstitution experiments in cell biology and related fields. It better mimics confinement conditions inside living cells, as opposed to conventional biochemical reconstitution. Methods for encapsulation inside GUVs are often not easy to implement, and success rates can differ significantly from lab to lab. One technique that has proven to be successful for encapsulating more complex protein systems is called continuous droplet interface crossing encapsulation (cDICE). Here, a cDICE-based method is presented for rapidly encapsulating cytoskeletal proteins in GUVs with high encapsulation efficiency. In this method, first, lipid-monolayer droplets are generated by emulsifying a protein solution of interest in a lipid/oil mixture. After being added into a rotating 3D-printed chamber, these lipid-monolayered droplets then pass through a second lipid monolayer at a water/oil interface inside the chamber to form GUVs that contain the protein system. This method simplifies the overall procedure of encapsulation within GUVs and speeds up the process, and thus allows us to confine and observe the dynamic evolution of network assembly inside lipid bilayer vesicles. This platform is handy for studying the mechanics of cytoskeleton-membrane interactions in confinement.

This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions.

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular ...