The authors thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for funding this work.

1. Preparation of Media and Cultures
<ol>
	<li>For LB + Amp: Prepare 500 ml of Luria-Bertani broth (LB) sterilized using an autoclave at 121 &#176;C for 30 min. Supplement with ampicillin to yield a final concentration of 100 &#181;g/ml of ampicillin and store at 4 &#176;C until required.</li>
	<li>For TB + Amp: Prepare for large (500 ml or greater) volumes of ministring production. Prepare 600 ml of sterile Terrific Broth (TB) sterilized using an autoclave at 121 &#176;C for 30 min. Supplement with ampicillin to yield a final concentration of 100 &#181;g/ml of ampicillin and store at 4 &#176;C until required.</li>
	<li>For LB agar + Amp: Prepare sterile LB agar plates by preparing LB + Amp supplemented with 15 g/L of agar. Aliquot approximately 15 - 20 ml in sterile Petri dishes and store upside down at 4 &#176;C until required.</li>
	<li>Prepare a streak plate of W3NN[pDMS] by aseptically streaking W3NN[pDMS] cells on LB agar + Amp. Incubate the plate overnight at 30 &#176;C, until single colonies can be observed. Do not incubate W3NN above this temperature to avoid derepressing protelomerase expression.</li>
	<li>Aseptically transfer 5 ml of LB + Amp into a sterile test tube. Prepare an overnight culture of W3NN[pDMS] by inoculating this volume with a single colony from the streak plate. Incubate the culture overnight at 30 &#176;C in a shaker at 230-250 rpm.</li>
</ol>
2. Growth Phase
<ol>
	<li>Aseptically transfer 10 ml of LB + Amp into a sterile 125 ml Erlenmeyer flask and inoculate with 100 &#181;l of the overnight W3NN[pDMS] culture into the prepared LB + Amp, making a 1:100 dilution.
	<ol>
		<li>For larger volumes, add 500 &#181;l of overnight culture into 50 ml of TB + Amp in a 250 ml Erlenmeyer flask, instead of LB + Amp.</li>
		<li>Alternatively, use as the second &#34;representative&#34; culture in step 2.2.</li>
	</ol>
	</li>
	<li>Incubate in a shaking incubator at 30 &#176;C and 250 rpm until the culture reaches mid to late log phase, indicated by an optical density (OD) or absorbance: A600 = 0.8. After 1-2 hr, the medium should begin to appear turbid to the naked eye.
	<ol>
		<li>Aseptically transfer 1 ml of culture to a clean spectrophotometer cuvette.</li>
		<li>Measure the light absorbance using a UV-vis spectrophotometer with the wavelength set to 600 nm. Use 1 ml of the media in which cells were grown to set the blank (LB or TB). Optionally, use the representative culture from step 2.1.3 for this step instead.</li>
		<li>If the culture has not yet reached the appropriate OD, return to the shaker and check once again every half hour. Check the OD more frequently as the A600 approaches 0.8.</li>
	</ol>
	</li>
	<li>Once the culture has reached late log phase, indicated by A600 = 0.8, aseptically transfer the culture into sterile 50 ml centrifuge tubes.</li>
	<li>Collect cells by centrifuging the culture at 10,000 x g for 10 min. Inspect for a pellet at the bottom of the tube.</li>
	<li>Decant the cleared supernatant into an appropriate biohazard waste container. Do not disturb the cell pellet.</li>
	<li>Repeat steps 2.4-2.5 until all cells have been collected.</li>
	<li>Re-suspend the pellet in 100 &#181;l LB + Amp. Aspirate using a micropipette or use a vortex mixer. For greater ministring production, re-suspend the pellet from a 50 ml TB + Amp culture into 1 ml of fresh LB + Amp media.</li>
</ol>
3. Ministring Production
<ol>
	<li>Aseptically add 20 ml of LB + Amp in a 125 ml Erlenmeyer flask and heat to 42 &#176;C. For greater ministring production, prepare 500 ml of LB + Amp, preheated to 42 &#176;C.</li>
	<li>Transfer the resuspension into the preheated medium.</li>
	<li>Incubate at 42 &#176;C and 250 rpm until past log phase, indicated by A600 = 1.0. Do not leave 20 ml of culture at 42 &#176;C past 1 hr.</li>
	<li>Reduce the temperature to 37 &#176;C and leave the culture for an additional 90 min. For 500 ml, leave the culture for an additional induction period of 90 - 120 min and do not decrease the temperature.</li>
	<li>Reduce the temperature to 30 &#176;C and leave the culture shaking at 200 rpm for an additional temperature downshift period of 2 hr. For 500 ml, extend the temperature downshift to 4 hr or overnight.</li>
</ol>
4. Harvesting Ministrings
<ol>
	<li>Aseptically transfer the culture into sterile 50 ml centrifuge tubes. Centrifuge at 10,000 x g for 10 min and inspect for a pellet.</li>
	<li>Decant the supernatant into an appropriate biohazard waste container without disturbing the pellet. Repeat until all the cells have been collected.</li>
	<li>Retain the pellet for plasmid extraction with any commercial extraction and endotoxin removal kit or flash freeze in liquid nitrogen and store at -80 &#176;C for later extraction.</li>
</ol>

<ol>
	<li>Klinman, D. M., Ylt, A. K., Beaucaget, S. L., Conover, J., Kriegt, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CpG+motifs+present+in+bacterial+DNA+rapidly+induce+lymphocytes+to+secrete+interleukin+6,+interleukin+12,+and+interferon+&#947;.">CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon &#947;.</a> Proc Natl Acad Sci. 93, 2879-2883 (1996).</li><li>Hodges, B. L., Taylor, K. M., Joseph, M. F., Bourgeois, S. A., Scheule, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+transgene+expression+from+plasmid+DNA+gene+therapy+vectors+is+negatively+affected+by+CpG+dinucleotides.">Long-term transgene expression from plasmid DNA gene therapy vectors is negatively affected by CpG dinucleotides.</a> Mol Ther. 10 (2), 269-278 (2004).</li><li>Darquet, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minicircle:+an+improved+DNA+molecule+for+in+vitro+and+in+vivo+gene+transfer.">Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer.</a> Gene Ther. 6 (2), 209-218 (1999).</li><li>Gracey Maniar, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minicircle+DNA+vectors+achieve+sustained+expression+reflected+by+active+chromatin+and+transcriptional+level.">Minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcriptional level.</a> Mol Ther. 21 (1), 131-138 (2012).</li><li>Catanese, D. J., Fogg, J. M., Ii, D. E. S., Gilbert, B. E., Zechiedrich, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercoiled+minivector+DNA+resists+shear+forces+associated+with+gene+therapy+delivery.">Supercoiled minivector DNA resists shear forces associated with gene therapy delivery.</a> Gene Ther. 19 (1), 94-100 (2011).</li><li>Hacein-bey-abina, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertional+oncogenesis+in+4+patients+after+retrovirus-mediated+gene+therapy+of+SCID-X1.">Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.</a> J Clin Invest. 118 (9), 3132-3142 (2008).</li><li>Nafissi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+ministrings:+highly+safe+and+effective+gene+delivery+vectors.">DNA ministrings: highly safe and effective gene delivery vectors.</a> Mol Ther Nucleic Acids. 3 (April), (2014).</li><li>Schakowski, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+minimal-size+vector+(MIDGE)+improves+transgene+expression+in+colon+carcinoma+cells+and+avoids+transfection+of+undesired+DNA.">A novel minimal-size vector (MIDGE) improves transgene expression in colon carcinoma cells and avoids transfection of undesired DNA.</a> Mol Ther. 3 (5 Pt 1), 793-800 (2001).</li><li>Wang, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+micro-linear+vector+for+in+vitro+and+in+vivo+gene+delivery+and+its+application+for+EBV+positive+tumors.">A novel micro-linear vector for in vitro and in vivo gene delivery and its application for EBV positive tumors.</a> PLoS One. 7 (10), (2012).</li><li>L&#243;pez-Fuertes, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccination+with+linear+minimalistic+(MIDGE)+vectors+confers+protection+against+Leishmania+major+infection+in+mice.">DNA vaccination with linear minimalistic (MIDGE) vectors confers protection against Leishmania major infection in mice.</a> Vaccine. 21 (3-4), 247-257 (2002).</li><li>Schakowski, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+size+MIDGE+vectors+improve+transgene+expression+in+vivo.">Minimal size MIDGE vectors improve transgene expression in vivo.</a> In Vivo. 21 (1), 17-23 (2007).</li><li>Hertwig, S., Klein, I., Lurz, R., Lanka, E., Appel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PY54,+a+linear+plasmid+prophage+of+Yersinia+enterocolitica+with+covalently+closed+ends.">PY54, a linear plasmid prophage of Yersinia enterocolitica with covalently closed ends.</a> Mol Microbiol. 48, 989-1003 (2003).</li><li>Nafissi, N., Slavcev, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+an+in-vivo+linear+covalently+closed+DNA+vector+production+system.">Construction and characterization of an in-vivo linear covalently closed DNA vector production system.</a> Microb Cell Fact. 11 (1), 154 (2012).</li><li>Xu, Z. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+transcriptional+regulatory+elements+for+constructing+plasmid+vectors.">Optimization of transcriptional regulatory elements for constructing plasmid vectors.</a> Gene. 272 (1-2), 149-156 (2001).</li><li>Hoffmann, F., Rinas, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+induced+by+recombinant+protein+production+in+Escherichia+coli.">Stress induced by recombinant protein production in Escherichia coli.</a> Adv Biochem Eng Biotechnol. 89, 73-92 (2004).</li><li>Sum, C., Chong, J. Y., Wettig, S., Slavcev, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+purification+of+linear+covalently+closed+deoxyribonucleic+acid+by+Q-anion+exchange+membrane+chromatography.">Separation and purification of linear covalently closed deoxyribonucleic acid by Q-anion exchange membrane chromatography.</a> J Chromatogr A. 1339, 214-218 (2014).</li><li>Hoffmann, F., Rinas, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmid+amplification+in+Escherichia+coli+after+temperature+upshift+is+impaired+by+induction+of+recombinant+protein+synthesis.">Plasmid amplification in Escherichia coli after temperature upshift is impaired by induction of recombinant protein synthesis.</a> Biotechnol Lett. 23, 1819-1825 (2001).</li><li>Nafissi, N., Sum, C. H., Wettig, S., Slavcev, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+one-step+heat-inducible+in+vivo+mini+DNA+vector+production+system.">Optimization of a one-step heat-inducible in vivo mini DNA vector production system.</a> PLoS One. 9 (2), (2014).</li></ol>

The authors declare they have no competing financial interests.

We describe here a simple system for the production of LCC DNA ministrings using a one-step heat-inducible protocol, which requires no special equipment aside from that which is used in typical bacterial growth and manipulation. DNA ministrings are torsion-free, stable linear DNA expression cassettes, devoid of prokaryotic genetic elements. They may be used in place of conventional methods for gene transfer, expression of reporter genes, as well as in assessments towards the efficiency of transgene expression in eukaryotic systems. DNA ministrings combine the biocompatibility and increased transfection efficiency benefits of &#34;mini&#34; vectors with the superior safety profile of linear DNA vectors. Our robust one-step in vivo production platform rapidly and easily produces DNA ministrings for any gene transfer application without the need for multiple costly in vitro processes.
There are several critical steps to ensure optimal ministring production (Figures 2 and 3). Heat induction is the most critical step to ministring production. Complete lack of ministring production is most likely due to lack of heat induction (cells retained at 30 &#176;C), where repression of protelomerase expression prevents conversion of precursor plasmid to LCC ministring. Following the heat induction protocol, expect a production efficiency of approximately 75%. Heat induction is optimal while cells are in log phase. Though there is no statistically significant difference in ministring PE when using cells in stationary phase (&#34;Overgrown&#34;, Figure 3), we did observe almost a 50% decrease in overall plasmid yield. Yields will depend on the plasmid extraction protocol used. For optimal ministring production, trigger heat induction while cells are in log phase. Poor ministring production will most likely be a result of prolonged temperature upshift or insufficient temperature downshift. Prolonged temperature upshift is discouraged as this activates the E. coli heat shock response15, which may inhibit protelomerase activity and interfere with plasmid stability. Therefore, timing the temperature downshift is another critical step for efficient ministring production. Without additional incubation time at 30 &#176;C, ministring production efficiency was found to be very poor (&#34;Early Removal&#34;, Figure 3). This may be attributed to the amount of time required for Tel expression and activity. The originating prophage PY54 encoding Tel protelomerase normally infects Yersinia, which optimally grows around 28 &#176;C12, indicating that 30 &#176;C may also be more optimal for Tel activity.
The DNA minivector production system utilizes an in vivo platform to generate high quality bacterial sequence-free DNA minivectors. Modifications to the protocol may include altering the growth media to optimize plasmid and protein production in order to promote cell growth during the growth phase (TB instead of LB), or increase ministring production and conversion efficiency. For larger culture volumes (200 ml and greater), temperature downshift and incubation at 30 &#176;C may be extended overnight without severe negative impact on ministring PE. We have included modifications in our protocol for 500 ml cultures as an example. Purification of DNA ministrings can be expedited through in vitro endonuclease digestion of the prokaryotic backbone. There are a number of endonuclease target sites (e.g., PI-SceI, PvuI, ScaI) available on the prokaryotic backbone of the precursor plasmid, which can be cut to expose open linear ends, allowing for in vitro degradation of the prokaryotic backbone. Isolation of ministrings from other vector species can also be achieved through anion exchange membrane chromatography16.
One current limitation associated with the LCC DNA vector production system is the need for purification of the DNA ministring from other LCC and CCC products. Although easily purified using standard plasmid isolation methods, the isolation step can still be a disadvantage in a large-scale setting. Separation of the ministring can be time-consuming using AGE if the ministring and the LCC prokaryotic backbone are too similar in size. Alternatively, anion exchange membrane chromatography may provide better separation of ministrings from CCC vector species16. Temperature upshift can be another potential limitation as high temperatures also activate the heat shock response in E. coli, which negatively affects recombinant protein yields17 and consequently reduce rates of plasmid to ministring conversion. We report elsewhere optimizations of the production platform to circumvent and/or mitigate such effects of heat shock18. We are also currently optimizing methods to eliminate or reduce LCC prokaryotic backbone contamination in vivo.
Chromosomal integration of the LCC DNA ministring disrupts the host chromosome and subjects the cell to apoptosis. Not only does this reduce potential negative consequences of insertional mutagenesis such as oncogenesis and silencing of adjacent genes, thereby improving its safety; the lethal effect can also serve as an ideal method for knock-in and gene replacement studies without the associated side effects and damage of integration. DNA ministring vectors can be applied towards the transfer and expression of genes for therapeutic proteins, RNA, antibodies, or antigens into a given target in vivo or replace a mal- or non- functional allele. The simple one-step heat-inducible in vivo production system allows DNA ministring vectors to be easily produced for clinical or industrial applications.

The goal is to produce scalable quantities of linear covalently closed (LCC) DNA minivectors using a simple and high efficiency one-step heat-inducible in vivo DNA ministring production system. DNA ministrings provide a safe and effective non-viral strategy to deliver DNA. They combine the safety of LCC vectors with the efficiency of DNA minicircles, and also offer a safer alternative to virus-derived vectors without compromising transfection efficiency.
In order for a transgene delivery system to be successful, the DNA vector must enter the target host cell and express the encoded transgene(s). There are several cellular barriers that need to be overcome in practice, particularly in mammalian systems. In order to avoid degradation by serum nucleases and immune detection by reticulo-endothelial system, the DNA vector must be bio- and immune-compatible with target system. Unprotected DNA is quickly digested by plasma nucleases and the plasmid membrane is composed of dense lipoprotein barriers so the DNA vector must be capable of rapidly crossing the plasma membrane of target cells. Once in the cell, vectors must traverse the cytoplasm and pass through the nuclear membrane to enter the nucleus for transgene expression. Non-viral gene delivery techniques focus on enhancement of tissue- and cell-targeting. However, the use of conventional plasmids with these techniques reduces transfection efficiency due to the presence of immunogenic bacterial sequences, which rapidly silences gene expression1,2. Conventional plasmids typically carry antibiotic resistance genes for maintenance in prokaryotic systems. However, these may produce potential adverse effects in human hosts or impart resistance to naturally occurring host flora via horizontal gene transfer effects. Conventional plasmids also contain dinucleotide CpG motifs2, which can trigger an unwanted immunostimulatory response, potentially reducing or silencing transgene expression.
As they are solely comprised of the eukaryotic expression cassette, DNA minivectors, such as DNA minicircles3, are a better alternative for gene delivery as they exhibit improved extracellular and intracellular bioavailability and improved gene expression due to their reduced size and absence of immunostimulatory prokaryotic elements4. The reduced vector size fares better with respect to resistance to shear forces associated with in vivo administration to a target site5. The higher copy number of the vector per unit mass requires less transfection reagent, thus decreasing toxicity. However, in the event of random vector integration into a host chromosome, circularly covalently closed (CCC) vectors, including both DNA minicircles and conventional plasmids, impart molecular continuity and therefore may lead to insertional mutagenesis, which can have devastating consequences6. Comparatively, integration of a linear DNA vector disrupts the chromosome and initiates cell death pathways, thereby removing the mutant cell from the proliferating cell population and preventing insertional mutagenesis7. Minimalistic immunologically defined gene expression (MIDGE) vectors8 and micro-linear vectors9 (MiLV) are LCC DNA vectors developed in vitro. MIDGE vectors have exhibited up to a 17-fold improved transgene expression in vivo compared to conventional plasmid DNA vectors11, and have demonstrated promising results in vaccine10 and cancer8 gene therapy. In addition, due to the torsion-free structure of the LCC minivector, less transfection reagent is required in comparison to the CCC supercoiled counterpart, thus reducing toxicity7.
Our enhanced LCC DNA vectors, DNA ministrings, have demonstrated superior expression efficiency and bioavailability7. Furthermore, DNA ministrings are produced on a one-step heat-inducible in vivo production system, an expedient and cost-effective alternative to MIDGE and MiLV LCC vectors, which require multiple steps in vitro. The DNA ministring production system to be demonstrated is a simple heat-inducible process performed in vivo, making it both cost-effective and easily scalable. This system exploits the Yersinia enterocolitica bacteriophage PY54-derived Tel/pal protelomerase recombination system12 to separate the minimal eukaryotic expression cassette from the prokaryotic plasmid backbone. We have engineered Escherichia coli cells (W3NN) to express Tel protelomerase under the control of the heat-inducible bacteriophage &#955; promoter, cI[Ts]85713. Upon expression, Tel protelomerase acts on pal target sites present within &#34;Super Sequence&#34; (SS) sites located on the precursor plasmid to yield LCC products, from which the DNA ministring can then be purified (Figure 1). The SS sites on the DNA ministring precursor plasmid also encode target sites for other recombinases including Cre recombinase (lox), TelN protelomerase (telRL) and Flp recombinase (FRT), thereby facilitating the production of isogenic LCC and CCC minivectors from one precursor plasmid. In addition, each SS site is flanked on both sides by SV40 enhancer (SV40e) sequences, which serve to improve nuclear translocation14. We have demonstrated elsewhere that successive addition of SV40e progressively confers corresponding increases in transfection efficiency7. The precursor plasmid (pDNA MiniString or pDMS) contains a polylinker (Figure 1) to facilitate insertion of any desired gene of interest in transcriptional fusion with the green fluorescent reporter (GFP).
The DNA minivector technology may be used in place of conventional methods for gene transfer, expression of reporter genes, and assessments towards the efficiency of transgene expression in eukaryotic systems. DNA ministrings combine the biocompatibility and increased transfection efficiency benefits of &#34;mini&#34; vectors with the superior safety profile of linear DNA vectors. Our robust one-step production platform rapidly and easily produces DNA ministrings for any gene transfer application and offers a higher safety profile.

<table border="0" cellpadding="0" cellspacing="0" width="1428">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">E. coli W3NN&#160;</td>
			<td style="width:295px;">Mediphage</td>
			<td style="width:268px;"></td>
			<td style="width:492px;">strain for ministring processing: Nafissi &#38; Slavcev, 2012</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">pDMS.IRES.GFP</td>
			<td style="width:295px;">Mediphage</td>
			<td style="width:268px;"></td>
			<td>parent plasmid for ministring processing: also referred to as pDNA Ministring (pDMS)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">BD&#8482; Difco&#8482; Dehydrated Culture Media: LB Broth, Miller (Luria-Bertani)</td>
			<td style="width:295px;">Fisher Scientific</td>
			<td>244620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Fisher BioReagents&#8482; Terrific Broth</td>
			<td style="width:295px;">Fisher Scientific</td>
			<td style="width:268px;">BP2468500</td>
			<td style="width:492px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">BD&#8482; Bacto&#8482; Dehydrated Agar</td>
			<td style="width:295px;">Fisher Scientific</td>
			<td style="width:268px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Ampicilin</td>
			<td style="width:295px;">MP Biomedicals LCC</td>
			<td style="width:268px;">2190147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Ultrapure Milipore Water</td>
			<td style="width:295px;">Fisher Scientific&#160;</td>
			<td style="width:268px;">Z00Q0V0US</td>
			<td>Model: Mili-Q Advantage A10 Water Purification System</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Surgical Gloves</td>
			<td style="width:295px;">VWR</td>
			<td style="width:268px;">(S) 82026-424, (M)82026-426, (L) 82026-428, (XL) 82026-430&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Fisherbrand&#8482; Petri Dishes with Clear Lid Raised Ridge (100 x 15 mm)</td>
			<td style="width:295px;">Fisher Scientific</td>
			<td style="width:268px;">FB0875712&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">30 mL KIMAX&#174; Test Tubes</td>
			<td style="width:295px;">VWR</td>
			<td style="width:268px;">89001-448</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Spectrophotometer Cuvette</td>
			<td style="width:295px;">Sarstedt</td>
			<td style="width:268px;">67.74</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sterile 15 mL Falcon Tubes</td>
			<td style="width:295px;">&#160;BD Falcon</td>
			<td>62406-200,</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">&#160;Sterile 50 mL Centrifuge Tubes</td>
			<td style="width:295px;">&#160;FroggaBio</td>
			<td>2930-S0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon&#8482; Disposable Polystyrene Serological Pipets 10 mL</td>
			<td style="width:295px;">BD Falcon</td>
			<td>13-675-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon&#8482; Disposable Polystyrene Serological Pipets 25 mL</td>
			<td style="width:295px;">BD Falcon</td>
			<td>13-668-2&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Micropipettor (100-1000 &#956;L)</td>
			<td style="width:295px;">FroggaBio</td>
			<td style="width:268px;">C1000-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Micropipettor (20-200 &#956;L)</td>
			<td style="width:295px;">FroggaBio</td>
			<td style="width:268px;">C200-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sterile Pipette Tips (100-1250 &#956;L)</td>
			<td style="width:295px;">VWR</td>
			<td style="width:268px;">89079-472&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sterile Pipette Tips (1-200 &#956;L)</td>
			<td style="width:295px;">VWR</td>
			<td style="width:268px;">89079-460</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Fisher Scientific Economy Burner</td>
			<td style="width:295px;">Fisher Scientific</td>
			<td style="width:268px;">03-917Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Incubator Shaker</td>
			<td style="width:295px;">New Brunswick Scientific&#160;</td>
			<td style="width:268px;">M1352-00000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sterile Erlenmeyer Flask (125 mL)</td>
			<td style="width:295px;">Pyrex (Sigma Aldrich)</td>
			<td>CLS4980125 Aldrich&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sterile Erlenmeyer Flask (250 mL)</td>
			<td style="width:295px;">Pyrex (Sigma Aldrich)</td>
			<td>CLS4980250 Aldrich&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">&#160;Sterile Erlenmeyer Flask (500 mL)</td>
			<td style="width:295px;">Pyrex (Sigma Aldrich)</td>
			<td>CLS4980500 Aldrich&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Spectrophotometer</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:268px;">335901</td>
			<td>Model: Genesys 10 Vis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Floor Centrifuge</td>
			<td style="width:295px;">Beckman Coulter&#160;</td>
			<td style="width:268px;">369001</td>
			<td>Model: Avanti J-E</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Benchtop Centrifuge</td>
			<td style="width:295px;">Beckman</td>
			<td style="width:268px;">362114</td>
			<td>Model: GS-6R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Fisher Scientific Analog Vortex Mixer&#160;</td>
			<td style="width:295px;">Fisher Scientific&#160;</td>
			<td style="width:268px;">02-215-365</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">1 kb DNA Ladder</td>
			<td style="width:295px;">New England BioLabs</td>
			<td style="width:268px;">N3232S</td>
			<td></td>
		</tr>
	</tbody>
</table>

production of double-stranded dna ministrings

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ministrings possess a heightened safety profile and also efficiently deliver DNA cargo to targeted cells. Conventional DNA vectors carry undesirable prokaryotic sequences, including antibiotic resistance genes, CpG motifs, and bacterial origins of replication, which may lead to the stimulation of host immunological responses. The bioavailability of conventional DNA vectors is also compromised due to their larger molecular size. Their circular nature may also impart chromosomal integration, leading to insertional mutagenesis.
Bacterial sequences are excised from DNA minivectors, leaving only the gene of interest (GOI) and necessary eukaryotic expression elements. Our LCC DNA minivectors, or DNA ministrings, are devoid of immunogenic bacterial sequences; therefore improving their bioavailability and GOI expression. In the event of vector integration into the chromosome, the LCC DNA ministring will lethally disrupt the host chromosome, thereby removing the potentially dangerous mutant from the proliferating cell population. Consequently, DNA ministrings offer the benefits of &#39;minicircle&#39; DNA while eliminating the potential for undesirable vector integration events. In comparison to conventional plasmids and their isogenic circular covalently closed (CCC) counterparts, DNA ministrings demonstrate superior bioavailability, transfection efficiency, and cytoplasmic kinetics - they thus require lower amounts of cationic surfactants for effective transfection of target cells.
We have constructed a one-step inducible in vivo system for the production of DNA ministrings in Escherichia coli that is simple to use, rapid, and scalable.

After DNA extraction, residual precursor plasmid, LCC prokaryotic backbone, and the DNA ministring will be recovered. Overall plasmid yield and purity can be assessed using a spectrophotometer. Purity is estimated using the ratio of the absorbance at 260 nm and 280 nm (A260/A280) where a ratio of 1.9-2.0 is optimal. All LCC and CCC products can be separated through agarose gel electrophoresis (AGE) (Figure 2). AGE visualizes the results of ministring production after heat induction, prior to ministring purification. A qualitative assessment of ministring production can be made at this stage by comparing band intensity of the ministring (2.4 kb) to parent plasmid (5.6 kb) (Figure 2). Ministring DNA can be recovered via standard plasmid gel extraction protocols following AGE. Figure 3 summarizes ministring production efficiencies under various conditions likely to occur while carrying out the protocol. Ministring production efficiency was determined based on DNA band intensity using the manufacturer&#39;s analysis program. Any similar densitometry program can be used to analyze the results of AGE.
<img alt="Figure 1" src="/files/ftp_upload/53177/53177fig1highres.jpg" width="700" /> 
Figure 1: Overview of ministring production. (A) The parent vector contains a polylinker in transcriptional fusion with a reporter gene, framed by the two SS sites. The backbone encodes beta-lactamase for ampicillin resistance and also contains several cut sites unique to the backbone for in vitro digestion. (B) Overview of the main protocol. Steps 2) Growth Phase and 3) Ministring Production are represented in a diagram to provide an overall schematic of the steps involved, highlighting the simplicity of the protocol. (C) Ministring induction. Heat induction inactivates the temperature sensitive repressor, allowing tel expression followed by Tel activity on SS sites in the parent plasmids. <a href="https://www.jove.com/files/ftp_upload/53177/53177fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53177/53177fig2highres.jpg" width="350" /> 
Figure 2: Ministring production after deviations from protocol. 1: Control, 2: Uninduced, 3: Overgrown, 4: Overinduced, 5: Early Removal. DNA ministring is approximately 2.4 kb, LCC backbone is 3 kb, and pNN9 parent plasmid is 5.6 kb. Band intensity of ministring and LCC backbone bands drop with deviations from protocol, indicating lower ministring yield. From left to right: 1 kb ladder from New England Biolabs; Control, following protocol; Uninduced, no temperature upshift; Overgrown, temperature upshift during stationary phase; Overinduced, extended temperature upshift; Early removal, no temperature downshift. Plasmids were extracted using the Omega-Biotek E.Z.N.A. Plasmid Mini Kit. <a href="https://www.jove.com/files/ftp_upload/53177/53177fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53177/53177fig3highres.jpg" width="550" /> 
Figure 3: Effects of deviations from protocol on ministring production efficiency (PE). Results are the mean of 3 trials. PE is estimated from relative band intensity measured using AlphaImager HP after AGE. The deviations occur at the following steps: Control, following protocol; Uninduced, no temperature upshift at step 2; Overgrown, temperature upshift began during stationary phase at step 1.6; Overinduced, extended temperature upshift at step 2.3; Early removal, cells removed and extracted without temperature downshift at step 2.3. <a href="https://www.jove.com/files/ftp_upload/53177/53177fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Production of Double-stranded DNA Ministrings at JoVE.com

Production of Double-stranded DNA Ministrings

The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings.

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ...

production-of-double-stranded-dna-ministrings

Research

JoVE Journal

Biology

10.3K Views.  University of Waterloo. The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings. 

Video: Production of Double-stranded DNA Ministrings

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication1. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures2. SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA3. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length4 and DNA ends (blunt or overhang)5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqA&Delta;(41-59)-A25R) to obtain crystals suitable for structure determination.

Crystal structure of protein&#x2013;DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...