Name: detection of live escherichia coli o157:h7 cells by pma-qpcr
Uploaded: 2014-02-01T14:00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR at JoVE.com

We thank the U.S. Department of Homeland Security for providing part of the funding.

1. Bacterial Strains and DNA Template Preparation
<ol>
	<li>Grow bacterial strains of E. coli O157:H7, non-O157, and other pathogenic species, such as Salmonella and Shigella, at 37 &#176;C overnight with shaking.</li>
	<li>Extract DNA from bacterial cultures using the appropriate kit (see table of materials) and following manufacturer&#39;s instructions.</li>
	<li>Measure optical density (OD260) to determine the DNA concentrations by using a spectrophotometer.</li>
</ol>
2. Primer and Probe Design for the qPCR Assay
<ol>
	<li>E. coli O157:H7 sequences are from GenBank accession number AE00517411. Design primers and probe using appropriate software for primer and probe design for real-time PCR (see materials table for details). 
	The sequences of primers and probe are as follows: Z3276-Forward (F) 5&#39;-TATTCCGCGATGCTTGTTTTT-3&#39; 
	Z3276-Reverse (R) 5&#39;- ATTATCTCACCAGCAAACTGGCGG-3&#39; 
	Z3276-Probe, FAM-CCGCAATCTTTCC- MGBNFQ</li>
	<li>Reconstitute the primer powder with water, resulting in a concentration of 10 &#956;M as a primer work solution.</li>
	<li>Dilute the probes to 10 &#956;M as probe work solutions. Labeled probes vary in potency with batches. Therefore, titrate each batch of labeled probes before using for optimal qPCR performance.</li>
</ol>
3. Setting the qPCR Assay Conditions
<ol>
	<li>Prepare reaction mixture, which consists of 25.0 &#956;l of 2x real-time PCR master mix, 200 nM of forward primer, 200 nM of reverse primer, and 100 nM of probe.</li>
	<li>Add 5 &#956;l of sample DNA (100 pg) and an appropriate volume of water to reach a final volume of 50 &#956;l.</li>
	<li>For nontemplate control, use 5 &#956;l of water to replace DNA sample.</li>
	<li>Set the qPCR conditions as follows: 95 &#176;C for 10 min for activation of TaqMan; followed by 40 cycles of 95 &#176;C for 10 sec for denaturation and 60 &#176;C for 1 min for annealing/extension.</li>
</ol>
4. qPCR Sensitivity Test
<ol>
	<li>Make a serial 10-fold dilution from a culture of mid-exponential phase (OD600 = 0.5; about 1.5 x 108 CFU/ml by plating).</li>
	<li>Add 100 ml of cell dilutions onto a 96-well plate in triplicate.</li>
	<li>Centrifuge the plate at 2,500 x g for 10 min.</li>
	<li>Add 50 ml of DNA extraction solution to each well.</li>
	<li>Resuspend cell pellets with a multi-channel pipette.</li>
	<li>Seal the plate with a film, boil the plate in a water bath for 10 min, and centrifuge at 2,500 x g for 2 min.</li>
</ol>
5. Preparing Live and Dead Cell Mixtures for PMA Treatment
<ol>
	<li>Grow 10 ml of E. coli O157:H7 at 37 &#176;C to mid-exponential phase and divide the culture into two aliquots.</li>
	<li>Boil one aliquot of cells for 10 min in a water bath for dead cells and leave the other aliquot for live cells.</li>
	<li>Verify there are no live cells from the heat-killed aliquot by plating the cells on LB agar plates.</li>
	<li>Take 2 ml of the live and heat-killed cells and adjust the concentration to 8 x 106 CFU/ml with LB medium. Create four sets of cell dilutions ranging from 8 x 100 to 8 x 106 CFU/ml.</li>
	<li>Use the first two sets of cell dilutions for live cells treated with PMA or untreated.</li>
	<li>To the third and fourth sets of cell dilutions, add 8 x 106 dead cells to each cell dilution (8 x 100&#160;-&#160;8 x 106) to make live and dead cell mixtures.</li>
	<li>Treat the third set of dilution with PMA and use the fourth set of dilution for control.</li>
</ol>
6. Treating Cells with PMA
<ol>
	<li>Dissolve PMA in dimethyl sulfoxide (or water) to make 10 mM stock solution and stored at -20 &#176;C in the dark.</li>
	<li>Aliquot 400 &#956;l of the live cells, dead cells, and mixture of live and dead cells in three separate microtubes.</li>
	<li>Add 2.0 ml of 10 mM PMA to each cell aliquot to a final concentration of 50 &#956;M.</li>
	<li>Incubate the samples at ambient temperature for 5 min in the dark, shaking gently a few times, 3 sec each time.</li>
	<li>Add 100 &#956;l of samples on a 96-well plate.</li>
	<li>Seal the plate with an optical film and put the plate on ice.</li>
	<li>Set the plate 20 cm from a 650 W halogen light source and expose for 2 min for light exposure.</li>
	<li>Centrifuge the plate at 2,500 x g for 10 min, gently discard the supernatant and carefully drain the plate on a piece of absorbing paper.</li>
	<li>Resuspend cell pellets in 50 &#956;l of DNA extraction solution by pipetting up and down twenty times with a multi-channel Pipetman.</li>
	<li>Seal the plate with a film, boil for 10 min in a water bath, and centrifuge at 2,500 x g for 2 min. The supernatant in the plate is the DNA from cells treated with PMA and ready for qPCR.</li>
	<li>Perform qPCR as described above, using 5 &#956;l of DNA.</li>
</ol>

<ol>
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The authors have nothing to disclose.

A primary aim of the study involves use of an ORF Z3276, a unique to E. coli O157:H7, as a sole biomarker for qPCR assay. At present, most qPCR assays are targeting virulence genes, such as stx1, stx2, and eaeA1,2,5,6 or shared phenotypic genes, such as uidA3,4, rfbE and fliC genes8,9. Occasionally, a species or strain cannot be definitely identified by the virulence gene(s), such as stx1and stx2 genes, as those genes are present in different species or strains16 as well. Using rfbE and fliC for target genes, both genes are needed to be used for complete identification17. Using ORF Z3276 as a biomarker, this qPCR assay has been demonstrated to be sensitive and specific assay (Table 1). This assay has been subjected to stringent inclusivity and exclusivity tests, including O157:H7 strains (n = 367), over 100 non-O157 strains, and other pathogenic species, such as Salmonella and Shigella. All O157:H7 strains examined were positively detected, and no cross-reactivity was found from the non-O157 strains (Table 1), indicating that ORF Z3276 is a unique and strong biomarker for detection of E. coli O157:H7.
The other objective of the study is to selectively detect live cells from dead cells by PMA treatment before DNA extraction. PMA can penetrate into the dead cells with compromised membrane and bind to DNA, and thus inhibits the DNA amplification of dead cells in PCR13. We have modified the PMA-treatment procedure, and adapted it to a 96-well plate format for the procedure. The right intensity of light exposure is essential to obtain the optimal cross-linking effect. Previously, microtubes have been used in this step13-15. However, separate microtubes are difficult to handle in light exposure step, and these tubes are not absolutely transparent to obtain the best cross-liking. Using a 96-well plate, we improved the efficiency of PMA-treatment. These changes may impact the assay in several ways: they make it easier to obtain a thorough and uniform cross-linking effect, especially with numerous samples; the treated samples can be moved from one plate to another to make it possible for detection of large number of samples; and they provide this PMA-qPCR assay with automation potential for the whole process. The limitation of this PMA-qPCR assay is that PMA treatment increases the PCR assay cost somewhat and slightly reduced the sensitivity of PCR assay.

PCR is a common technique for detection of E. coli O157:H7 from food and environmental samples. Identifying specific biomarkers for E. coli O157:H7 is one of the key factors for successful detection in PCR assays. The biomarkers commonly used in PCR assays for detection of E. coli O157:H7 include Shiga toxins (stx1 and stx2)1,2, uidA3,4, eaeA5,6, rfbE7, and fliC genes8,9. These biomarkers can provide variable efficiency for identification; however, most of the biomarkers cannot be used as a unique biomarker for detection of E. coli O157:H7. This inadequacy in differentiation power of target genes calls for more specific and stronger biomarker(s) for identification of E. coli O157:H710.
Numerous probes for genes or hypothetical open reading frames (ORFs) in E. coli O157:H711 were designed for qPCR assay based on the clues from DNA genotyping microarrays from our laboratory and by searching within the GenBank database of the National Center for Biotechnology Information. The sequence of Z3276 ORF, which is a fimbrial gene11, was selected for qPCR assay. Subsequently, a qPCR assay was developed through numerous trials on PCR parameters such as concentrations of primers and probes, as well as annealing and extension temperatures (data not shown). After incentive inclusivity and exclusivity tests on reference strains such as E. coli O157:H7, non-O157 and Shigella strains in the qPCR, the ORF Z3276 was identified as a specific and strong genetic marker for identification of E. coli O157:H7. Then we develop a new qPCR method using this unique biomarker for identification of E. coli O157:H7.
Another challenge in detection of E. coli O157:H7 in contaminated foods and environment is accurate determination of live cells from samples. The extended presence of DNA from dead cells and inability to differentiate viability in PCR assay could cause false-positive readings, resulting in overestimation of live cell numbers in detection. Consequently, this limits the PCR usage in accurate detection of foodborne pathogens12. A novel approach to differentiate DNA of the dead cells has been taken by combining propidium monoazide (PMA) treatment with PCR procedure in the last few years13-15. PMA is able to go inside of dead cells, and intercalate into the DNA when exposed to light, but it cannot go inside of live cells to react with the DNA of the live cells. Thus, in PMA-treated mixtures of live and dead cells, the DNA of dead cells cannot be amplified in PCR13. We combined PMA treatment with the newly developed qPCR assay to selectively detect live E. coli O157:H7 cells. Additionally, we have made the PMA-qPCR assay possible for high throughput detection.

<table border="0" cellpadding="0" cellspacing="0" width="901">
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		<col />
		<col />
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	<tbody>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">AB 7900HT Fast Real-time PCR system</td>
			<td style="width:164px;">ABI</td>
			<td style="width:171px;"></td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">2 x Universal master mix</td>
			<td style="width:164px;">ABI</td>
			<td align="right" style="width:171px;">4304437</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">PrepMan Ultra</td>
			<td style="width:164px;">ABI</td>
			<td align="right" style="width:171px;">4318930</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Primer Express</td>
			<td style="width:164px;">ABI</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="58" rowspan="2" style="height:58px;width:249px;">Probes</td>
			<td rowspan="2" style="width:164px;">ABI</td>
			<td>Top of Form</td>
			<td rowspan="2"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">4316033 Bottom of Form</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">PMA</td>
			<td style="width:164px;">Biodium</td>
			<td align="right" style="width:171px;">40013</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="87" rowspan="3" style="height:87px;width:249px;">Puregen cell and tissue kit</td>
			<td rowspan="3" style="width:164px;">Qiagene</td>
			<td>Top of Form</td>
			<td rowspan="3"></td>
		</tr>
		<tr height="29">
			<td align="right" height="29" style="height:29px;">158622</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">Bottom of Form</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">DU530 spectrophotometer</td>
			<td style="width:164px;">Beckman</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Spectrophotometer</td>
			<td style="width:164px;">NanoDrop Technology</td>
			<td style="width:171px;">&#160;ND-1000</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">650 w halogen light source</td>
			<td style="width:164px;">Britek</td>
			<td style="width:171px;">H8061S</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Otptical Adhesive film</td>
			<td>ABI</td>
			<td align="right">4311971</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Optical 96-well reaction plate</td>
			<td>ABI</td>
			<td align="right">4316813</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of live escherichia coli o157:h7 cells by pma-qpcr

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of E. coli O157:H7 by qPCR using ORF Z3276
One of the key factors of this assay is the sensitivity and specificity of the Z3276 probe used in the qPCR. It can detect as low as a few copies of the genome of DNA of E. coli O157:H7 as shown in Figure 1A. Of the 120 non-O157 strains examined, including the six major non-O157 STEC strains, O104:H4 strains, Salmonella, and Shigella strains, no cross-reactivity was found, as shown in Table 1.
Combination of the PMA treatment with qPCR
Incubating E. coli O157:H7 cells with PMA (50 mM) for 5 min in the dark and exposing to light for 2 min were selected for PMA treatment conditions. Additionally, we modified PMA treatment procedure. Compared with various transparent microtubes used in the cross-linking step, a 96-well plate was found to be more efficient to reach the optimal cross-linking effect and more convenient than handling a large number of sample tubes during the light exposure process.
Amplification of DNA from PMA-treated live and dead cells in qPCR
The effects of PMA-mediated inhibition of amplification of DNA from dead cells on this qPCR assay were shown in Figure 1 by using DNA derived from two serial 10-fold live or dead cell dilutions. The results show that the curves generated from the live cells treated with PMA (red curve) and without PMA (blue curve) seem linear and nearly identical to one another. Slight CT value differences were shown between the live cells treated with PMA and without PMA (Figure 1A), suggesting that PMA treatment had little effect on amplification of DNA of the live cells in the qPCR. But a 15-CT value difference (32,000 fold) was shown between the dead cells treated with PMA and without PMA, demonstrating that PMA treatment efficiently suppressed DNA amplification from the dead cells (Figure 1B).
Detection of live cells from live and dead cell mixtures in qPCR
Two sets of live cell dilutions (8 x 100&#160;-&#160;8 x 106 CFU) were treated with PMA or without PMA to detect live cells from live/dead cell mixtures. The qPCR results (Figure 2A) demonstrated a parallel inverse progressive trend of CT values as related to the numbers of treated live cells (purple bars) to the untreated samples (blue bars). The treated live cells yielded a somewhat higher CT values than those of untreated live cells. A similar inverse progressive trend in CT values was observed with the real number of live cells in the live/dead cell mixtures treated with PMA (green bars) as well in Figure 2B. In addition, this downward trend of CT values was observed with the number of live cells in the mixtures despite the presence of 106 dead cells. These results showed that the CT values of the live/dead cell mixtures represented the DNA from the live cells alone, whereas the DNA amplification from the dead cells was efficiently suppressed by PMA treatment. But, the CT values of live/dead cell mixtures treated with PMA were fluctuated and failed to reflected the live cell numbers in the mixtures in Figure 2B (yellow bars).
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50967/50967fig1highres.jpg" src="/files/ftp_upload/50967/50967fig1.jpg" /> 
Figure 1. Live and dead cells treated with PMA or without PMA in qPCR assay. (A) A serial of 10-fold-diluted live E. coli O157:H7 cell dilutions (8 x 100 - 8 x 106 CFU) were treated with PMA or without PMA. The CT values represent the average CT values of the triplicates in the qPCR assay. (B) Comparison of DNA amplification of live cells and dead cells treated with PMA and without PMA in the q comparison qPCR assay. &#916;Rn refers to fluorescence intensity change. <a href="https://www.jove.com/files/ftp_upload/50967/50967fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50967/50967fig2highres.jpg" src="/files/ftp_upload/50967/50967fig2.jpg" /> 
Figure 2. Differentiation of live cells from live/dead cell mixtures by PMA-qPCR. Four sets of 10-fold dilutions of cell cultures (8 x 100 - 8 x 106 CFU) were made as indicated. (A) First set of cell dilution was treated with PMA while the second cell dilution was or untreated before DNA extraction. (B) 8 x 106 dead cells were mixed with the third and fourth sets of cell dilutions to make two sets of live/dead cell mixtures as indicated. One set of the cell mixtures was treated with PMA and the other set was treated without PMA before DNA extraction. Each bar represents the average CT values of a triplicate experiment &#177; SD.
<table>
	<tbody>
		<tr>
			<td>Organism</td>
			<td>Serotype</td>
			<td>No. of strains tested</td>
		</tr>
		<tr>
			<td rowspan="15">E. coli</td>
			<td>2006 spinach O157:H7 outbreak isolates</td>
			<td>186</td>
		</tr>
		<tr>
			<td>2006 Taco Bell O157:H7 isolates</td>
			<td>58</td>
		</tr>
		<tr>
			<td>2006 Taco John O157:H7 isolates</td>
			<td>11</td>
		</tr>
		<tr>
			<td>DMB strain collections from other O157:H7 outbreaks</td>
			<td>112</td>
		</tr>
		<tr>
			<td>O104:H4</td>
			<td>3</td>
		</tr>
		<tr>
			<td>O26</td>
			<td>18</td>
		</tr>
		<tr>
			<td>O103</td>
			<td>13</td>
		</tr>
		<tr>
			<td>O111</td>
			<td>24</td>
		</tr>
		<tr>
			<td>O121</td>
			<td>2</td>
		</tr>
		<tr>
			<td>O45</td>
			<td>5</td>
		</tr>
		<tr>
			<td>O145</td>
			<td>9</td>
		</tr>
		<tr>
			<td>O113</td>
			<td>2</td>
		</tr>
		<tr>
			<td>O91</td>
			<td>1</td>
		</tr>
		<tr>
			<td>O55</td>
			<td>9</td>
		</tr>
		<tr>
			<td>Other</td>
			<td>19</td>
		</tr>
		<tr>
			<td rowspan="2">Salmonella</td>
			<td>Typhi</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Newport</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Shigella dysenteriae</td>
			<td>6</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Shigella sonnei</td>
			<td>Unknown</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Shigella flexneri</td>
			<td>4</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Shigella boydii</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td colspan="2">Total</td>
			<td>481</td>
		</tr>
	</tbody>
</table>
Table 1. Strains used for validation in real-time PCR assay for identification of E. coli O157:H7.

Watch this Scientific Journal Video about Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR at JoVE.com

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

detection-of-live-escherichia-coli-o157h7-cells-by-pma-qpcr

Research

JoVE Journal

Biology

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

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 ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.